Diagnosis of melanoma and solar lentigo by nucleic acid analysis

ABSTRACT

The present invention provides methods for diagnosing melanoma and/or solar lentigo in a subject by analyzing nucleic acid molecules obtained from the subject. The present invention also provides methods for distinguishing melanoma from solar lentigo and/or dysplastic nevi and/or normal pigmented skin. The methods include analyzing expression or mutations in epidermal samples, of one or more skin markers. The methods can include the use of a microarray to analyze gene or protein profiles from a sample

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/172,784 filedFeb. 4, 2014 which is a continuation of U.S. application Ser. No.12/991,685 filed Mar. 14, 2011, which is a U.S National Stageapplication of International Application No. PCT/US2009/044035 filed May14, 2009, which claims the benefit under 35 USC §119(e) to U.S.Application Ser. No. 61/058,149 filed Jun. 2, 2008, U.S. ApplicationSer. No. 61/053,998 filed May 16, 2008 and U.S. Application Ser. No.61/127,731 filed May 14, 2008. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

FIELD OF THE INVENTION

The invention relates generally to methods of characterizing pigmentedskin lesions suspected of being melanomas using primarily non-invasiveskin sampling.

BACKGROUND INFORMATION

Melanoma is a serious form of skin cancer in humans. It arises from thepigment cells (melanocytes), usually in the skin. The incidence ofmelanoma is increasing at the fastest rate of all cancers in the UnitedStates with a lifetime risk of 1 in 68. Although melanoma accounts foronly 4% of all dermatologic cancers, it is responsible for 80% of alldeaths from skin cancers. It has long been realized that recognition anddiagnosis of melanoma, when it is early stage disease, is key to itscure.

Given this, it is imperative that research be carried out not only ontherapeutics for melanoma, but also on all aspects of melanoma includingprevention and detection. Most of these deaths from melanoma could havebeen prevented if the melanomas, initially located on the skin, couldhave been detected in their early stages. The ability to cure melanomain its earliest skin stage, in situ, is virtually 100% if the melanomais adequately surgically excised. If the melanoma is caught in a laterstage, where it has invaded to a depth of 4 mm or more, the ten-yearsurvival rate is less than 50%. If the melanoma is not detected until ithas spread to distant parts of the body (Stage IV), the prognosis isdismal, with only 7-9% of patients surviving 5 years, with the mediansurvival time being 8-9 months. The long-term “cure” rate for Stage IVmelanoma is only 1-2%.

To advance early detection of melanoma, several things must be improved.People need to be better educated with regards to the risks of melanomaand how to prevent and detect it on their own skin. Also physicians needto be more alert to the possibility of melanoma and be better trained indetection. But even if these two areas are improved, the diagnosis ofmelanoma on the skin is still difficult. Studies have shown that evenexpert clinicians working in pigmented lesion clinics where melanoma istheir specialty are only able to determine whether a suspiciouspigmented lesion is melanoma or not with 60-80% sensitivity. This leadsto the need for surgical biopsy of large numbers of pigmented lesionsfor every melanoma that is detected, and, doubtless, to the missing ofsome melanomas in their early stages.

In current practice melanoma is diagnosed by biopsy andhistopathological examination; approximately 20 to 30 biopsies must beperformed to find one melanoma and even then some melanomas are missedin the earliest stage. The limitations of visual detection are apparentto dermatologists who are constantly searching for ways to betterdetermine whether suspicious lesions are melanoma or not without havingto cut them out first. To this end, epiluminescence microscopy (ELM) hascome into use. This is a method whereby lesions are looked at using adevice that simultaneously magnifies the lesion while reducing visualinterference from refractive index differences at the skin-airinterface. While ELM does give a different view, it is of limitedimprovement. Studies have shown that until one becomes fairly skilled inutilizing the instrument, sensitivity in detection of melanoma actuallydecreases. Even very skilled users of ELM improve their ability todetect melanomas only by 5-10%. This still leads to an unacceptablesensitivity in detection and the need to biopsy large numbers of benignlesions to detect a few melanomas. And again, some melanomas will bemissed completely in their early stages.

Clearly there is a need for further development of technology that willenable physicians to determine the nature and extent of suspiciouslesions of the skin. Such technology would ideally directly assay thephysiology of the suspect lesion to enable a sensitive diagnosis.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that analysisof nucleic acid molecules or of protein expression products of nucleicacid molecules from specific genes can be used to characterize skinlesions in a subject. The method provides valuable genetic informationbased on DNA, messenger RNA, or protein expression products obtainedtherefrom, for example.

In one embodiment, the method involves use of a non-invasive approachfor recovering nucleic acids such as DNA or messenger RNA or proteinsfrom the surface of skin via a tape stripping procedure that permits adirect quantitative and qualitative assessment of biomarkers. Althoughtape-harvested nucleic acid and protein expression products are shown tobe comparable in quality and utility to recovering such molecules bybiopsy, the non-invasive method provides information regarding cells ofthe outermost layers of the skin that may not be obtained using biopsysamples. Finally, the non-invasive method is far less traumatic than abiopsy.

Thus, the non-invasive method is used to capture cells on pigmented skinlesions that are suspected of being melanomas. Nucleic acid moleculesobtained from skin cells captured by the non-invasive method areanalyzed in order to diagnose the nature of the lesion (e.g., malignantmelanoma). In one embodiment, a nucleic acid molecule is amplified priorto analysis. Secondary outcomes could include tests for diagnosis andprognosis of a variety of pigmented skin lesions and even to predict atherapeutic regimen. In another embodiment, the skin cells are lysed toextract one or more proteins, which are then quantitated to diagnose thenature of the lesion. It should be understood that the methods of theinvention are not limited to non-invasive techniques for obtaining skinsamples. For example, but not by limitation, one of skill in the artwould know other techniques for obtaining a skin sample such as scrapingof the skin, biopsy, suction, blowing and other techniques. As describedherein, non-invasive tape stripping is an illustrative example forobtaining a skin sample.

In another embodiment, the methods involve detection of one or moremutations in the nucleic acid sequence of the nucleic acid moleculeobtained from the skin. Such mutations may be a substitution, adeletion, and/or an insertion of the nucleic acid sequence that resultsin a diseased state in the subject from which the skin sample isobtained.

In one embodiment, the nucleic acid molecule analyzed is listed inTables 10-12 and 15. In another embodiment, the method further includesanalyzing one or more nucleic acid molecules listed Tables 1-8. Forexample, in one embodiment, the gene analyzed is any one or more ofinterferon regulatory factor 6, claudin 23, melan-A, osteopetrosisassociated transmembrane protein 1, RAS-like family 11 member B, actininalpha 4, transmembrane protein 68, Glycine-rich protein (GRP3S),Transcription factor 4, hypothetical protein FLJ20489, cytochrome csomatic, transcription factor 4, Forkhead box P1, transducer of ERBB2-2,glutaminyl-peptide cyclotransferase (glutaminyl cyclase), hypotheticalprotein FLJ10770, selenophosphate synthetase 2, embryonal Fyn-associatedsubstrate, Kruppel-like factor 8, Discs large homolog 5 (Drosophila),regulator of G-protein signalling 10, ADP-ribosylation factor relatedprotein 2, TIMP metallopeptidase inhibitor2,5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMPcyclohydrolase, similar to RIKEN cDNA 5730421E18 gene, Regulator ofG-protein signalling 10, Nuclear RNA-binding protein putative,tyrosinase-related protein 1, TIMP metallopeptidase inhibitor 2, Claudin1, transcription factor 4, solute carrier family 16 (monocarboxylic acidtransporters) member 6 (similar to solute carrier family 16 member 6;monocarboxylate transporter 6), or any combination thereof. In anotherembodiment, the nucleic acid molecule is from one or more genes listedin Tables 10-12 and 15.

Accordingly, provided herein is a method for characterizing and/ordiagnosing melanoma in a subject, including obtaining a nucleic acidmolecule or protein by biopsy of a skin lesion on the subject, andanalyzing the nucleic acid molecule to distinguish melanoma fromdysplastic nevi and/or normal pigmented skin in the subject. In thismethod, at least one nucleic acid molecule whose expression isinformative of melanoma is detected in the epidermal sample. In oneexample, expression of one or more of the genes listed in Tables 1-8,10-12, 15, or a combination thereof, is detected in the epidermal sampleto characterize the melanoma. In one embodiment, the gene is any one ormore of interferon regulatory factor 6, claudin 23, melan-A,osteopetrosis associated transmembrane protein 1, RAS-like family 11member B, actinin alpha 4, transmembrane protein 68, Glycine-richprotein (GRP3S), Transcription factor 4, hypothetical protein FLJ20489,cytochrome c somatic, transcription factor 4, Forkhead box P1,transducer of ERBB2-2, glutaminyl-peptide cyclotransferase (glutaminylcyclase), hypothetical protein FLJ10770, selenophosphate synthetase 2,embryonal Fyn-associated substrate, Kruppel-like factor 8, Discs largehomolog 5 (Drosophila), regulator of G-protein signalling 10,ADP-ribosylation factor related protein 2, TIMP metallopeptidaseinhibitor 2,5-aminoimidazole-4-carboxamide ribonucleotideformyltransferase/IMP cyclohydrolase, similar to RIKEN cDNA 5730421E18gene, Regulator of G-protein signalling 10, Nuclear RNA-binding proteinputative, tyrosinase-related protein 1, TIMP metallopeptidase inhibitor2, Claudin 1, transcription factor 4, solute carrier family 16(monocarboxylic acid transporters) member 6 (similar to solute carrierfamily 16 member 6; monocarboxylate transporter 6), or any combinationthereof.

The non-invasive methods of the invention involve applying an adhesivetape to a target area of skin in a manner sufficient to isolate a sampleadhering to the adhesive tape, wherein the sample includes nucleic acidmolecules or proteins. Typically, at least one nucleic acid molecule orprotein whose expression is informative of melanoma is detected in thesample. The method of characterizing skin using tape stripping has anumber of applications, such as the following: (i) diseaseclassification/subclassification; (ii) monitoring disease severity andprogression; (iii) monitoring treatment efficacy; and (iv) prediction ofa particular treatment regimen. All of these applications, whichthemselves represent embodiments disclosed herein, preferably usenon-invasive sampling to recover information that is otherwise difficultor impractical to recover (e.g., through the use of biopsies). Theinformation may be contained in the DNA, protein, or RNA of skin cellsclose to the surface of the skin. In one embodiment, expression of oneor more of the genes listed in Tables 1-8, 10-12, 15, or a combinationthereof, is detected in the sample to characterize the sample. Thisexemplary method is particularly useful for distinguishing melanoma fromdysplastic nevi and/or normal pigmented skin. In one embodiment,expression of one or more of the genes listed in Table 12 or 15 isdetected in the sample to characterize the sample.

As such, also provided herein is a method for distinguishing solarlentigines from dysplastic nevi and/or basal cell carcinoma and/ornormal pigmented skin in a subject, including applying an adhesive tapeto a target area of skin in a manner sufficient to isolate a sampleadhering to the adhesive tape, wherein the sample includes nucleic acidmolecules. At least one nucleic acid molecule whose expression isinformative of solar lentigo is detected in the sample. In oneembodiment, expression of one or more of the genes listed in Tables10-12, 15, or a combination thereof, is detected in the sample tocharacterize the melanoma. In another embodiment, expression of one ormore of the genes listed in Table 12 or 15 is detected in the sample tocharacterize the solar lentigo.

Other embodiments are based in part on the discovery that for tapestripping of the skin, non-polar, pliable, adhesive tapes, especiallypliable tapes with rubber adhesive, are more effective than other typesof adhesive tapes. In some embodiments, the tape comprises a rubberadhesive on a polyurethane film. Using pliable tapes with rubberadhesives, as few as 10 or less tape strippings and in certain examplesas few as 4 or even 1 tape stripping can be used to isolate and/ordetect nucleic acid molecules from the epidermal layer of the skin.

In another embodiment, the methods of the invention provide forcharacterization of a skin lesion in situ, including application of adetectably labeled probe directly to a skin lesion for visual analysis.At least one nucleic acid molecule whose expression is informative ofmelanoma or dysplastic nevi or normal skin is detected on the skinlesion or surrounding margin or tissue using a specific probe. In oneexample, expression of one or more of the genes listed in Tables 1-8,10-12, 15, or a combination thereof, is detected on the skin lesion orsurrounding margin or tissue to characterize the melanoma. In oneembodiment, expression of one or more of the genes listed in Tables10-12 or 15 is detected in the sample to characterize the melanoma.

Also provided herein is a method for diagnosing a disease state byestablishing a gene expression pattern of a target area suspected ofbeing melanoma on the skin of a subject and comparing the subject's geneexpression profile to a reference gene expression profile obtained froma corresponding normal skin sample. In one embodiment, the target areaof the skin simultaneously expresses a plurality of genes at the proteinlevel that are markers for melanoma. In another embodiment, the genesare listed in Tables 1-8, 10-12, 15, or any combination thereof. Inanother embodiment, the genes are listed in Tables 8 or 12.

In one embodiment, the method of diagnosing a disease state involvesdetection of one or more mutations in the nucleic acid sequence of thegene. Such mutations may be a substitution, a deletion, and/or aninsertion of the nucleic acid sequence that results in a diseased statein the subject from which the skin sample is obtained. In oneembodiment, the genes are listed in Tables 1-8, 10-12, 15, or anycombination thereof. In another embodiment, the genes are listed inTables 8 or 12.

In another aspect, the invention provides kits for characterizing a skinlesion in a subject. In one embodiment, the kit includes a skin samplecollection device, such as a biopsy needle or an adhesive tape fornon-invasive tape stripping, and one or more probes or primers thatselectively bind to one or more nucleic acid molecules in any of Tables1-8 and 10-12, 15, or to a nucleic acid or protein expression product ofa nucleic acid molecule in any of Tables 1-8, 10-12, and 15. Forexample, in one embodiment, the gene analyzed is any one or more ofinterferon regulatory factor 6, claudin 23, melan-A, osteopetrosisassociated transmembrane protein 1, RAS-like family 11 member B, actininalpha 4, transmembrane protein 68, Glycine-rich protein (GRP3S),Transcription factor 4, hypothetical protein FLJ20489, cytochrome csomatic, transcription factor 4, Forkhead box P1, transducer of ERBB2-2,glutaminyl-peptide cyclotransferase (glutaminyl cyclase), hypotheticalprotein FLJ10770, selenophosphate synthetase 2, embryonal Fyn-associatedsubstrate, Kruppel-like factor 8, Discs large homolog 5 (Drosophila),regulator of G-protein signalling 10, ADP-ribosylation factor relatedprotein 2, TIMP metallopeptidase inhibitor2,5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMPcyclohydrolase, similar to RIKEN cDNA 5730421E18 gene, Regulator ofG-protein signalling 10, Nuclear RNA-binding protein putative,tyrosinase-related protein 1, TIMP metallopeptidase inhibitor 2, Claudin1, transcription factor 4, solute carrier family 16 (monocarboxylic acidtransporters) member 6 (similar to solute carrier family 16 member 6;monocarboxylate transporter 6), or any combination thereof. In anotherembodiment, the kit includes a microarray containing at least a fragmentof a gene or a nucleic acid or protein product of a gene identified inany of Tables 1-8, 10-12, 15, or any combination thereof.

In another embodiment, the kit for characterizing a skin lesion in asubject includes an applicator and one or more probes or primers thatselectively bind to one or more nucleic acid molecules in any of Tables1-8 and 10-12, 15, or to a nucleic acid or protein expression product ofa nucleic acid molecule in any of Tables 1-8, 10-12, and 15. In oneembodiment, the probes are detectably labeled for visual identificationof expression of RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are graphical diagrams showing data from the EDR, PTP,and PTN as a function of sample size, assuming a threshold for declaringthe significance of a probe/gene expression difference between nevi andprimary melanoma of p<0.05.

FIGS. 2A and 2B are graphical diagrams showing data from a sample sizeanalysis that considered the contrast results for nevi vs. primarymelanoma in the context of an analysis of variance (ANOVA) comparingnormal skin, nevi, and primary melanoma

FIGS. 3A and 3B are graphical diagrams showing data from an analysisfocusing exclusively on the posterior true probability (PTP) fordifferent assumed significance levels.

FIGS. 4A to 4D are pictorial and graphical diagrams showing thedevelopment of a gene classifier for distinguishing melanoma fromatypical nevi and normal pigmented skin.

FIGS. 5A and 5B are graphical diagrams showing data from predictionanalysis of the developed classifiers for distinguishing melanoma fromatypical nevi and normal pigmented skin.

FIGS. 6A to 6E are graphical diagrams showing data from predictionanalysis of the developed classifiers for distinguishing melanoma fromatypical nevi and normal pigmented skin.

FIG. 7 is a hierarchial cluster analysis of the identified genesdistinguishing melanoma from atypical nevi and normal pigmented skin.

FIG. 8 is a graphical diagram showing results from classificationmodeling of the identified genes.

FIG. 9 is a graphical diagram showing data of a developed classifier fordistinguishing melanoma from atypical nevi and normal pigmented skin.

FIG. 10 is a pictorial diagram showing the development of a classifierto discriminate melanoma from atypical nevi using non-invasive tapestrip-based genomic profiling.

FIG. 11 is a pictorial diagram describing the development of a 19-geneclassifier that discriminates melanoma from atypical nevi.

FIG. 12 is a pictorial diagram showing a hierarchial cluster analysis ofthe identified genes from the 19-gene classifier identified in FIG. 11.

FIG. 13 is a pictorial diagram showing results from 10 melanoma and 10nevi samples against the 19-gene classifier identified in FIG. 11.

FIG. 14 is a graphical diagram showing data of a developed classifierfor distinguishing solar lentigines from normal pigmented skin.

FIG. 15 is a hierarchial cluster analysis of the identified genes fromFIG. 14 distinguishing solar lentigines from normal pigmented skin.

FIG. 16 is a graphical diagram showing data from prediction analysis ofthe developed classifiers for distinguishing solar lentigines fromnormal pigmented skin.

FIG. 17 is a hierarchial cluster analysis of a gene expression profiledistinguishing solar lentigines from atypical nevi and basal cellcarcinoma.

FIG. 18 is a hierarchial cluster analysis of a gene expression profiledistinguishing solar lentigines from lentigo maligna.

FIG. 19 is a hierarchial cluster analysis of a 28-gene classifierdistinguishing solar lentigines from lentigo maligna.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery that analysisof nucleic acid molecules or of protein expression products of nucleicacid molecules from specific genes can be used to characterize skinlesions in a subject. Accordingly, the present invention providesmethods and kits useful for detecting cancer, especially melanoma, bydetermining the expression profiles of one or more specific genes ofinterest. In addition, the present invention provides methods and kitsuseful for distinguishing solar lentigines from cancer by determiningthe expression profiles of one or more specific genes of interest.

There are two main motivations for conducting genome wide expressionprofiling studies in melanoma. First, melanoma is one of the bestcharacterized carcinogenesis models for gradual progression of benignlesions to cancer: normal pigmented cells to nevi to atypical nevi toprimary melanoma in situ to invasive primary melanoma to aggressivemetastatic melanoma. This progression is known to correlate withdistinctive chromosomal changes, and is thought to be mediated bystepwise progressive changes in gene expression, suggesting thatexpression profiling may identify genes responsible for tumorigenesis inmelanoma. Indeed, candidate tumor genes have been identified withmicroarray analyses of melanoma cell lines. The second reason is thatmolecular characterization of tumors may allow a better stagingclassification of tumors and prognosis prediction. While histologicalcharacteristics such as the thickness and ulceration of tumors have somevalue as predictors of prognosis, there is lack of informative markersthat help determine which patients will do well and which patients willhave progressive disease and metastasis. Molecular markers identified inmicroarray experiments of tumors are already being introduced intoclinical practice in the management of breast cancer. Gene expressionprofiling experiments in melanoma and melanoma cell lines suggest thatthe classification of melanoma can be improved, but studies are lackingwith sufficient power to define molecular criteria for diagnosis oridentify prognostic markers; the establishments of such markers wouldrepresent a major advance in melanoma care. A major reason for the lackof powerful microarray studies in melanoma is that, unlike most solidtumors, it is necessary to paraffin embed and section the whole lesionfor histology, leaving no sample for RNA isolation. Although thissituation is now changing, the ability to avoid biopsy until adefinitive diagnosis is made would be powerful for subjects that wouldnot normally be eligible for one or more biopsies.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

The term “subject” as used herein refers to any individual or patient towhich the subject methods are performed. Generally the subject is human,although as will be appreciated by those in the art, the subject may bean animal. Thus other animals, including mammals such as rodents(including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits,farm animals including cows, horses, goats, sheep, pigs, etc., andprimates (including monkeys, chimpanzees, orangutans and gorillas) areincluded within the definition of subject.

As used herein, the terms “sample” and “biological sample” refer to anysample suitable for the methods provided by the present invention. Asample of cells can be any sample, including, for example, a skin sampleobtained by non-invasive tape stripping or biopsy of a subject, or asample of the subject's bodily fluid. Thus, in one embodiment, thebiological sample of the present invention is a tissue sample, e.g., abiopsy specimen such as samples from needle biopsy. In one embodiment,the term “sample” refers to any preparation derived from skin of asubject. For example, a sample of cells obtained using the non-invasivemethod described herein can be used to isolate nucleic acid molecules orproteins for the methods of the present invention. Samples for thepresent invention typically are taken from a skin lesion, which issuspected of being the result of a disease or a pathological orphysiological state, such as psoriasis or dermatitis, or the surroundingmargin or tissue. As used herein, “surrounding margin” or “surroundingtissue” refers to tissue of the subject that is adjacent to the skinlesion, but otherwise appears to be normal or free from lesion.

As used herein “corresponding normal cells” or “corresponding normalsample” refers to cells or a sample from a subject that is from the sameorgan and of the same type as the cells being examined. In one aspect,the corresponding normal cells comprise a sample of cells obtained froma healthy individual that does not have a skin lesion or skin cancer.Such corresponding normal cells can, but need not be, from an individualthat is age-matched and/or of the same sex as the individual providingthe cells being examined. Thus, the term “normal sample” or “controlsample” refers to any sample taken from a subject of similar speciesthat is considered healthy or otherwise not suffering from theparticular disease, pathological or physiological state, or from thesame subject in an area free from skin lesions. As such, anormal/standard level of RNA denotes the level of RNA present in asample from the normal sample. A normal level of RNA can be establishedby combining skin samples or cell extracts taken from normal healthysubjects and determining the level of one or more RNAs present. Inaddition, a normal level of RNA also can be determined as an averagevalue taken from a population of subjects that is considered to behealthy, or is at least free of a particular disease, pathological orphysiological state. Accordingly, levels of RNA in subject, control, anddisease samples can be compared with the standard values. Deviationbetween standard and subject values establishes the parameters fordiagnosing or characterizing disease.

The term “skin” refers to the outer protective covering of the body,consisting of the epidermis (including the stratum corneum) and theunderlying dermis, and is understood to include sweat and sebaceousglands, as well as hair follicle structures. Throughout the presentapplication, the adjective “cutaneous” can be used, and should beunderstood to refer generally to attributes of the skin, as appropriateto the context in which they are used. The epidermis of the human skincomprises several distinct layers of skin tissue. The deepest layer isthe stratum basalis layer, which consists of columnar cells. Theoverlying layer is the stratum spinosum, which is composed of polyhedralcells. Cells pushed up from the stratum spinosum are flattened andsynthesize keratohyalin granules to form the stratum granulosum layer.As these cells move outward, they lose their nuclei, and thekeratohyalin granules fuse and mingle with tonofibrils. This forms aclear layer called the stratum lucidum. The cells of the stratum lucidumare closely packed. As the cells move up from the stratum lucidum, theybecome compressed into many layers of opaque squamae. These cells areall flattened remnants of cells that have become completely filled withkeratin and have lost all other internal structure, including nuclei.These squamae constitute the outer layer of the epidermis, the stratumcorneum. At the bottom of the stratum corneum, the cells are closelycompacted and adhere to each other strongly, but higher in the stratumthey become loosely packed, and eventually flake away at the surface.

As used herein, the term “skin lesion” refers to a change in the coloror texture in an area of skin. As such, “skin lesions suspected of beingmelanoma” are skin lesions with characteristics of malignant melanoma,which are well known to those of skill in the art, such asdermatologists and oncologists. Such lesions are sometimes raised andcan have a color that is different from the color of normal skin of anindividual (e.g., brown, black, red, or blue). Lesions suspected ofbeing melanoma sometimes include a mixture of colors, are oftenasymmetrical, can change in appearance over time, and may bleed. A skinlesion suspected of being melanoma may be a mole or nevus. Melanomalesions are usually, but not always, larger than 6 mm in diameter.Melanoma includes superficial spreading melanoma, nodular melanoma,acral lentiginous melanoma, and lentigo maligna melanoma. The term“lentigo maligna” refers to a precancerous lesion on the skin,especially in areas exposed to the sun, that is flat, mottled, andbrownish with an irregular outline and grows slowly over a period ofyears. Melanoma can occur on skin that has been overexposed to the sun.Therefore, in one embodiment the skin sample is taken from an area ofskin that has been overexposed to the sun.

The term “dysplastic nevus” refers to an atypical mole or a mole whoseappearance is different from that of common moles. Dysplastic nevi aregenerally larger than ordinary moles and have irregular and indistinctborders. Their color frequently is not uniform and ranges from pink todark brown; they usually are flat, but parts may be raised above theskin surface. Dysplastic naevus can be found anywhere, but are mostcommon on the trunk of a subject.

The term “cancer” as used herein, includes any malignant tumorincluding, but not limited to, carcinoma and sarcoma. Cancer arises fromthe uncontrolled and/or abnormal division of cells that then invade anddestroy the surrounding tissues. As used herein, “proliferating” and“proliferation” refer to cells undergoing mitosis. As used herein,“metastasis” refers to the distant spread of a malignant tumor from itssight of origin. Cancer cells may metastasize through the bloodstream,through the lymphatic system, across body cavities, or any combinationthereof. The term “cancerous cell” as provided herein, includes a cellafflicted by any one of the cancerous conditions provided herein. Theterm “carcinoma” refers to a malignant new growth made up of epithelialcells tending to infiltrate surrounding tissues, and to give rise tometastases. The term “melanoma” refers to a malignant tumor ofmelanocytes which are found predominantly in skin but also in bowel andthe eye. “Melanocytes” refer to cells located in the bottom layer, thebasal lamina, of the skin's epidermis and in the middle layer of theeye. Thus, “melanoma metastasis” refers to the spread of melanoma cellsto regional lymph nodes and/or distant organs (e.g., liver, brain,breast, prostate, etc.).

The term “basal cell carcinoma” or “BCC” refers to a slow-growingneoplasm that is locally invasive but rarely metastasizes. It is derivedfrom basal cells, the deepest layer of epithelial cells of the epidermisor hair follicles. BCC is a common skin cancer that is often associatedwith overexposure to sunlight.

The term “solar lentigo” or “solar lentigines,” also known as asun-induced freckle or senile lentigo, is a dark (hyperpigmented) lesioncaused by natural or artificial ultraviolet (UV) light. Solar lentiginesmay be single or multiple. Solar lentigines are benign, but they doindicate excessive sun exposure, a risk factor for the development ofskin cancer. The lesions tend to increase in number with age, makingthem common among the middle age and older population. They can vary insize from about 0.2 to 2.0 cm. These flat lesions usually have discreteborders, are dark in color, and have an irregular shape.

As used herein, the term “gene” refers to a linear sequence ofnucleotides along a segment of DNA that provides the coded instructionsfor synthesis of RNA, which, when translated into protein, leads to theexpression of hereditary character. As such, the term “skin marker” or“biomarker” refers to a gene whose expression level is different betweenskin surface samples at the site of malignant melanoma and skin surfacesamples of normal skin or a lesion, which is benign, such as a benignnevus. Therefore, expression of a melanoma skin marker of the inventionis related to, or indicative of, melanoma. Many statistical techniquesare known in the art, which can be used to determine whether astatistically significant difference in expression is observed at a high(e.g., 90% or 95%) confidence level. As such, an increase or decrease inexpression of these genes is related to and can characterize malignantmelanoma. In one embodiment, there is at least a two-fold difference inlevels between skin sample near the site of malignant melanoma and skinsamples from normal skin.

As used herein, the term “nucleic acid molecule” means DNA, RNA,single-stranded, double-stranded or triple stranded and any chemicalmodifications thereof. Virtually any modification of the nucleic acid iscontemplated. A “nucleic acid molecule” can be of almost any length,from 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275,300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000,30,000, 40,000, 50,000, 75,000, 100,000, 150,000, 200,000, 500,000,1,000,000, 1,500,000, 2,000,000, 5,000,000 or even more bases in length,up to a full-length chromosomal DNA molecule. For methods that analyzeexpression of a gene, the nucleic acid isolated from a sample istypically RNA.

Micro-RNAs (miRNA) are small single stranded RNA molecules an average of22 nucleotides long that are involved in regulating mRNA expression indiverse species including humans (reviewed in Bartel 2004). The firstreport of miRNA was that of the lin-4 gene, discovered in the worm C.elegans (Lee, Feinbaum et al. 1993). Since then hundreds of miRNAs havebeen discovered in flies, plants and mammals. miRNAs regulate geneexpression by binding to the 3′-untranslated regions of mRNA andcatalyze either i) cleavage of the mRNA; or 2) repression oftranslation. The regulation of gene expression by miRNAs is central tomany biological processes such as cell development, differentiation,communication, and apoptosis (Reinhart, Slack et al. 2000; Baehrecke2003; Brennecke, Hipfner et al. 2003; Chen, Li et al. 2004). Recently ithas been shown that miRNA are active during embryogenesis of the mouseepithelium and play a significant role in skin morphogenesis (Yi,O'Carroll et al. 2006).

Given the role of miRNA in gene expression it is clear that miRNAs willinfluence, if not completely specify the relative amounts of mRNA inparticular cell types and thus determine a particular gene expressionprofile (i.e., a population of specific mRNAs) in different cell types.In addition, it is likely that the particular distribution of specificmiRNAs in a cell will also be distinctive in different cell types. Thus,determination of the miRNA profile of a tissue may be used as a tool forexpression profiling of the actual mRNA population in that tissue.Accordingly, miRNA levels and/or detection of miRNA mutations are usefulfor the purposes of disease detection, diagnosis, prognosis, ortreatment-related decisions (i.e., indicate response either before orafter a treatment regimen has commenced) or characterization of aparticular disease in the subject.

As used herein, the term “protein” refers to at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. A protein may be made up of naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures. Thus “amino acid”, or “peptide residue”, as used hereinmeans both naturally occurring and synthetic amino acids. For example,homo-phenylalanine, citrulline and noreleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesimino acid residues such as proline and hydroxyproline. The side chainsmay be in either the (R) or the (S) configuration.

A “probe” or “probe nucleic acid molecule” is a nucleic acid moleculethat is at least partially single-stranded, and that is at leastpartially complementary, or at least partially substantiallycomplementary, to a sequence of interest. A probe can be RNA, DNA, or acombination of both RNA and DNA. It is also within the scope of thepresent invention to have probe nucleic acid molecules comprisingnucleic acids in which the backbone sugar is other that ribose ordeoxyribose. Probe nucleic acids can also be peptide nucleic acids. Aprobe can comprise nucleolytic-activity resistant linkages or detectablelabels, and can be operably linked to other moieties, for example apeptide.

A single-stranded nucleic acid molecule is “complementary” to anothersingle-stranded nucleic acid molecule when it can base-pair (hybridize)with all or a portion of the other nucleic acid molecule to form adouble helix (double-stranded nucleic acid molecule), based on theability of guanine (G) to base pair with cytosine (C) and adenine (A) tobase pair with thymine (T) or uridine (U). For example, the nucleotidesequence 5′-TATAC-3′ is complementary to the nucleotide sequence5′-GTATA-3′.

The term “antibody” as used in this invention is meant to include intactmolecules of polyclonal or monoclonal antibodies, as well as fragmentsthereof, such as Fab and F(ab′)₂, Fv and SCA fragments which are capableof binding an epitopic determinant. The term “specifically binds” or“specifically interacts,” when used in reference to an antibody meansthat an interaction of the antibody and a particular epitope has adissociation constant of at least about 1×10⁻⁶, generally at least about1×10⁻⁷, usually at least about 1×10⁻⁸, and particularly at least about1×10⁻⁹ or 1×10⁻¹⁰ or less.

As used herein “hybridization” refers to the process by which a nucleicacid strand joins with a complementary strand through base pairing.Hybridization reactions can be sensitive and selective so that aparticular sequence of interest can be identified even in samples inwhich it is present at low concentrations. In an in vitro situation,suitably stringent conditions can be defined by, for example, theconcentrations of salt or formamide in the prehybridization andhybridization solutions, or by the hybridization temperature, and arewell known in the art. In particular, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature. For example,hybridization under high stringency conditions could occur in about 50%formamide at about 37° C. to 42° C. Hybridization could occur underreduced stringency conditions in about 35% to 25% formamide at about 30°C. to 35° C. In particular, hybridization could occur under highstringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS, and200 mg/ml sheared and denatured salmon sperm DNA. Hybridization couldoccur under reduced stringency conditions as described above, but in 35%formamide at a reduced temperature of 35° C. The temperature rangecorresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

As used herein, the term “mutation” refers to a change in the genomewith respect to the standard wild-type sequence. Mutations can bedeletions, insertions, or rearrangements of nucleic acid sequences at aposition in the genome, or they can be single base changes at a positionin the genome, referred to as “point mutations.” Mutations can beinherited, or they can occur in one or more cells during the lifespan ofan individual.

As used herein, the term “kit” or “research kit” refers to a collectionof products that are used to perform a biological research reaction,procedure, or synthesis, such as, for example, a detection, assay,separation, purification, etc., which are typically shipped together,usually within a common packaging, to an end user.

As used herein, the term “ameliorating” or “treating” means that theclinical signs and/or the symptoms associated with the cancer ormelanoma are lessened as a result of the actions performed. The signs orsymptoms to be monitored will be characteristic of a particular canceror melanoma and will be well known to the skilled clinician, as will themethods for monitoring the signs and conditions. Thus, a “treatmentregimen” refers to any systematic plan or course for treating a diseaseor cancer in a subject.

Samples from a tissue can be isolated by any number of means well knownin the art. Invasive methods for isolating a sample include, but are notlimited to the use of needles or scalpels, for example during biopsiesof various tissues. Non-invasive methods for isolating a sample include,but are not limited to tape-stripping and skin scraping.

Accordingly, in one embodiment, the present invention employs anon-invasive tape stripping technology to obtain samples of suspiciouslesions. As such, DNA microarray assays are used to create anon-invasive diagnostic for melanoma and/or distinguishing melanoma fromsolar lentigo. Tape-stripping removes superficial cells from the surfaceof the skin as well as adnexal cells. Small amounts of nucleic acidmolecules isolated from tape-stripped cells can be amplified and usedfor microarray analyses and quantitative PCR. In addition, proteinsobtained from the lysed cells may be quantitated for diagnosis ofdisease. Consequently, tape-stripping is a non-invasive diagnosticmethod, which does not interfere with subsequent histological analyses,thereby bypassing a major limitation to current expression profilingstudies on melanoma. While tape stripping will primarily samplesuperficial cells from the epidermis, this method holds great promise inthe diagnoses and prognosis prediction in pigmented lesions for thefollowing reasons: First, in contrast to benign nevi, in many melanomasthe pigmented cells migrate into the epidermis and/or adnexa.Consequently, this feature may help differentiate benign pigmentedlesions from melanomas based on tape stripping. Second, there arechanges in the dermis and epidermis adjacent to melanoma. The epidermalhyperplasia overlying melanoma seems to correlate with both angiogenesisand metastatic potential; these changes are expected to be sampled withthe tape stripping method. Finally, some advanced melanomas do reach thesurface of the skin and melanoma cancer cells would be sampled directlyby the tape stripping. In addition tape stripping is useful in the careof patients with multiple pigmented lesions where it is unpractical tobiopsy each and every lesion. Accordingly, the present inventiondemonstrates that stratum corneum RNA, harvested by tape stripping withEpidermal Genetic Information Retrieval (EGIR) (see U.S. Pat. No.6,949,338, incorporated herein by reference), can be used to distinguishmelanoma from dysplastic nevi in suspicious pigmented lesions.

As indicated, the tape stripping methods provided herein typicallyinvolve applying an adhesive tape to the skin of a subject and removingthe adhesive tape from the skin of the subject one or more times. Incertain examples, the adhesive tape is applied to the skin and removedfrom the skin about one to ten times. Alternatively, about ten adhesivetapes can be sequentially applied to the skin and removed from the skin.These adhesive tapes are then combined for further analysis.Accordingly, an adhesive tape can be applied to and removed from atarget site 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time, and/or 10, 9, 8, 7,6, 5, 4, 3, 2, or 1 adhesive tape can be applied to and removed from thetarget site. In one illustrative example, the adhesive tape is appliedto the skin between about one and eight times, in another example,between one and five times, and in another illustrative example the tapeis applied and removed from the skin four times.

The rubber based adhesive can be, for example, a synthetic rubber-basedadhesive. The rubber based adhesive in illustrative examples, has highpeel, high shear, and high tack. For example, the rubber based adhesivecan have a peak force tack that is at least 25%, 50%, or 100% greaterthan the peak force tack of an acrylic-based tape such as D-SQUAME™.D-SQUAME™ has been found to have a peak force of 2 Newtons, wherein peakforce of the rubber based adhesive used for methods provided herein, canbe 4 Newtons or greater. Furthermore, the rubber based adhesive can haveadhesion that is greater than 2 times, 5 times, or 10 times that ofacrylic based tape. For example, D-SQUAME™ has been found to haveadhesion of 0.0006 Newton meters, whereas the rubber based tape providedherein can have an adhesion of about 0.01 Newton meters using a textureanalyzer. Furthermore, in certain illustrative examples, the adhesiveused in the methods provided herein has higher peel, shear and tack thanother rubber adhesives, especially those used for medical applicationand Duct tape.

Virtually any size and/or shape of adhesive tape and target skin sitesize and shape can be used and analyzed, respectively, by the methods ofthe present invention. For example, adhesive tape can be fabricated intocircular discs of diameter between 10 millimeters and 100 millimeters,for example between 15 and 25 millimeters in diameter. The adhesive tapecan have a surface area of between about 50 mm² and 1000 mm², betweenabout 100 mm² to 500 mm² or about 250 mm².

In another embodiment, the sample is obtained by means of an invasiveprocedure, such as biopsy. Biopsies may be taken instead of or aftertape stripping and are subjected to standard histopathologic analysis.Analysis of biopsy samples taken simultaneously with tape strippingsamples may then be correlated with the data generated from one or moreof analysis of selected lesion RNA samples by DNA microarray,correlation of gene expression data with histopathology, and creation ofa candidate expression classifier for diagnosis of melanoma.

As used herein, “biopsy” refers to the removal of cells or tissues foranalysis. There are many different types of biopsy procedures known inthe art. The most common types include: (1) incisional biopsy, in whichonly a sample of tissue is removed; (2) excisional biopsy, in which anentire lump or suspicious area is removed; and (3) needle biopsy, inwhich a sample of tissue or fluid is removed with a needle. When a wideneedle is used, the procedure is called a core biopsy. When a thinneedle is used, the procedure is called a fine-needle aspiration biopsy.Other types of biopsy procedures include, but are not limited to, shavebiopsy, punch biopsy, curettage biopsy, and in situ biopsy. In anotherembodiment, the skin sample is obtained by scraping the skin with aninstrument to remove one or more nucleic acid molecules from the skin.

The skin sample obtained using the tape stripping method includes,epidermal cells including cells comprising adnexal structures. Incertain illustrative examples, the sample includes predominantlyepidermal cells, or even exclusively epidermal cells. The epidermisconsists predominantly of keratinocytes (>90%), which differentiate fromthe basal layer, moving outward through various layers having decreasinglevels of cellular organization, to become the cornified cells of thestratum corneum layer. Renewal of the epidermis occurs every 20-30 daysin uninvolved skin. Other cell types present in the epidermis includemelanocytes, Langerhans cells, and Merkel cells. As illustrated in theExamples herein, the tape stripping method of the present invention isparticularly effective at isolating epidermal samples.

Nucleic acid molecules can also be isolated by lysing the cells andcellular material collected from the skin sample by any number of meanswell known to those skilled in the art. For example, a number ofcommercial products available for isolating polynucleotides, includingbut not limited to, RNeasy™ (Qiagen, Valencia, Calif.) and TriReagent™(Molecular Research Center, Inc, Cincinnati, Ohio) can be used. Theisolated polynucleotides can then be tested or assayed for particularnucleic acid sequences, including a polynucleotide encoding a cytokine.Methods of recovering a target nucleic acid molecule within a nucleicacid sample are well known in the art, and can include microarrayanalysis.

Nucleic acid molecules may be analyzed in any number of ways known inthe art. For example, the presence of nucleic acid molecules can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments of the specific nucleic acid molecule. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the nucleic acid sequences to detect transformantscontaining the specific DNA or RNA.

In one embodiment, analysis of the nucleic acid molecules includesgenetic analysis is to determine the nucleotide sequence of a gene.Since a difference in length or sequence between DNA fragments isolatedfrom a sample and those of known sequences are due to an insertion,deletion, or substitution of one or more nucleotides, the determinationof nucleic acid sequences provides information concerning mutationswhich have absolute influence on the physiology of the disease state inthe subject. These mutations may also include transposition or inversionand are difficult to detect by other techniques than direct sequencing.For example, it has recently been shown that the presence of thec-kit-activating mutation, L576P, is indicative of malignant melanomas(see Table 1). Accordingly, the methods of the present invention may beused to detect genetic mutations in one or more genes listed in Tables1-8 and 10-12 for diagnosis and/or characterization of a skin lesion ina subject.

A variety of protocols for detecting and measuring the expression ofnucleic acid molecules, using either polyclonal or monoclonal antibodiesspecific for the protein expression product are known in the art.Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

In another embodiment, antibodies that specifically bind the expressionproducts of the nucleic acid molecules of the invention may be used tocharacterize the skin lesion of the subject. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule.

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to the nucleic acid molecules of Tables 1-8,10-12, and 15 include oligolabeling, nick translation, end-labeling orPCR amplification using a labeled nucleotide. Alternatively, the nucleicacid molecules, or any fragments thereof, may be cloned into a vectorfor the production of an mRNA probe. Such vectors are known in the art,are commercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland,Ohio). Suitable reporter molecules or labels, which may be used for easeof detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

PCR systems usually use two amplification primers and an additionalamplicon-specific, fluorogenic hybridization probe that specificallybinds to a site within the amplicon. The probe can include one or morefluorescence label moieties. For example, the probe can be labeled withtwo fluorescent dyes: 1) a 6-carboxy-fluorescein (FAM), located at the5′-end, which serves as reporter, and 2) a6-carboxy-tetramethyl-rhodamine (TAMRA), located at the 3′-end, whichserves as a quencher. When amplification occurs, the 5′-3′ exonucleaseactivity of the Taq DNA polymerase cleaves the reporter from the probeduring the extension phase, thus releasing it from the quencher. Theresulting increase in fluorescence emission of the reporter dye ismonitored during the PCR process and represents the number of DNAfragments generated. In situ PCR may be utilized for the directlocalization and visualization of target nucleic acid molecules and maybe further useful in correlating expression with histopathologicalfinding.

Means for producing specific hybridization probes for nucleic acidmolecules of the invention include the cloning of the nucleic acidsequences into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as ³²P or ³⁵S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

In order to provide a basis for the diagnosis or characterization ofdisease associated with expression of the nucleic acid molecules of theinvention, a normal or standard profile for expression is established.Standard hybridization may be quantified by comparing the valuesobtained from subjects of known skin characterization (e.g., fromsubjects either having melanoma, having dysplastic nevi, and/or havingsolar lentigines). Standard values obtained from such samples may becompared with values obtained from samples from subjects having skinlesions that are suspected of being melanoma. Deviation between standardand subject values is used to establish the presence of disease.

Accordingly, in one aspect of the invention, a non-invasive samplingmethod is provided for the characterization of skin lesion on the skin.In one embodiment, a sample set of pigmented skin lesions is created.Each sample consists of nucleic acid molecules recovered by tapestripping or biopsy sample of the superficial epidermis overlying thelesion. In addition to tape striping, a standard biopsy of the samelesion may also be performed, along with accompanying histology anddiagnosis. Nucleic acid molecules recovered by tape stripping thesuperficial epidermis of normal skin will serve as a negative control.

In another aspect, the invention provides a method of distinguishingmelanoma from solar lentigo and/or dysplastic nevi and/or normalpigmented skin in a subject. In one embodiment, the method includesanalyzing a nucleic acid molecule from one or more genes listed in anyof Tables 1-8, 10-12, 15, or any combination thereof. A target area ofthe skin of a subject that suspected of being melanoma is assayed forexpression of a large number of genes. Analyzing expression includes anyqualitative or quantitative method for detecting expression of a gene,many of which are known in the art. The method can include analyzingexpression of specific markers by measuring expression of the markersusing a quantitative method, or by using a qualitative method.Non-limiting methods for analyzing polynucleotides and polypeptides arediscussed below.

In another aspect, the invention provides a method of distinguishingsolar lentigines from dysplastic nevi and/or basal cell carcinoma and/ornormal pigmented skin in a subject. In one embodiment, the methodincludes analyzing a nucleic acid molecule from one or more genes listedin any of Tables 1-8, 10-12, 15, or any combination thereof. A targetarea of the skin of a subject that suspected of being melanoma isassayed for expression of a large number of genes. Analyzing expressionincludes any qualitative or quantitative method for detecting expressionof a gene, many of which are known in the art. The method can includeanalyzing expression of specific markers by measuring expression of themarkers using a quantitative method, or by using a qualitative method.Non-limiting methods for analyzing polynucleotides and polypeptides arediscussed below

Methods of analyzing expression of a gene of the present invention canutilize a microarray, or other miniature high-throughput technology, fordetecting expression of one or more gene products. Quantitativemeasurement of expression levels using such microarrays is also known inthe art, and typically involves a modified version of a traditionalmethod for measuring expression as described herein. For example, suchquantitation can be performed by measuring a phosphor image of aradioactive-labeled probe binding to a spot of a microarray, using aphosphor imager and imaging software.

In a related aspect, the invention provides a method for diagnosingvarious disease states in a subject by identifying new diagnosticmarkers, specifically the classification and diagnosis of melanoma. Inaddition, the invention provides a method for distinguishing solarlentigines from dysplastic nevi and/or lentigo maligna and/or normalskin. Thus, the invention provides a method for diagnosing variousdisease states in a subject by identifying new diagnostic markers,specifically the classification and diagnosis of melanoma. Byidentifying gene sets that are unique to a given state, thesedifferences in the genetic expression can be utilized for diagnosticpurposes. In one embodiment, the nucleic acid molecule is RNA, includingmessenger RNA (mRNA) that is isolated from a sample from the subject.Up-regulated and down-regulated gene sets for a given disease state maybe subsequently combined. The combination enables those of skill in theart to identify gene sets or panels that are unique to a given diseasestate. Such gene sets are of immense diagnostic value as they can beroutinely used in assays that are simpler than microarray analysis (forexample “real-time” quantitative PCR). Such gene sets also provideinsights into pathogenesis and targets for the design of new drugs.

A reference database containing a number of reference projected profilesis also created from skin samples of subjects with known states, such asnormal (i.e., non-melanoma) and various skin cancer disease statesand/or pigmented non-cancer states. The projected profile is thencompared with the reference database containing the reference projectedprofiles. If the projected profile of the subject matches best with theprofile of a particular disease state in the database, the subject isdiagnosed as having such disease state. Various computer systems andsoftware can be utilized for implementing the analytical methods of thisinvention and are apparent to one of skill in the art. Exemplarysoftware programs include, but are not limited to, Cluster & TreeView(Stanford, URLs: rana.lbl.gov or microarray.org), GeneCluster(MIT/Whitehead Institute, URL: MPR/GeneCluster/GeneCluster.html), ArrayExplorer (SpotFire Inc, URL: spotfire.com/products/scicomp.asp#SAE) andGeneSpring (Silicon Genetics Inc, URL:sigenetics.com/Products/GeneSpring/index.html) (for computer systems andsoftware, see also U.S. Pat. No. 6,203,987, incorporated herein byreference).

In another aspect, the methods of the present invention involve in situanalysis of the skin lesion for characterization thereof. For in situmethods, nucleic acid molecules do not need to be isolated from thesubject prior to analysis. In one embodiment, detectably labeled probesare contacted with a cell or tissue of a subject for visual detection ofexpressed RNA to characterize the skin lesion.

In another aspect, the methods of the present invention can also beuseful for monitoring the progression of diseases and the effectivenessof treatments. For example, by comparing the projected profile prior totreatment with the profile after treatment. In one embodiment, themethod characterizes a cancer as melanoma metastasis based on analysisof one or more nucleic acid molecules from Tables 1-8. In anotherembodiment, the method characterizes a solar lentigo based on analysisof one or more nucleic acid molecules from Tables 10-12 and 15. It isknown that in many cases, by the time a diagnosis of melanoma isestablished in a subject, metastasis has already occurred sincemelanomas contain multiple cell populations characterized by diversegrowth rates, karyotypes, cell-surface properties, antigenicity,immunogenicity, invasion, metastasis, and sensitivity to cytotoxic drugsor biologic agents. Thus, the present invention may be used tocharacterize cancer of an organ as having metastasized from melanoma.

In a related aspect, the methods of the present invention can also beuseful for determining an appropriate treatment regimen for a subjecthaving a specific cancer or melanoma. In another related aspect, themethods of the present invention can also be useful for determining anappropriate treatment regimen for a subject having solar lentigo. Thus,the methods of the invention are useful for providing a means forpracticing personalized medicine, wherein treatment is tailored to asubject based on the particular characteristics of the cancer or skinlesion in the subject. The method can be practiced, for example, byfirst determining whether the skin lesion is melanoma or solar lentigo,as described above.

The sample of cells examined according to the present method can beobtained from the subject to be treated, or can be cells of anestablished cancer cell line of the same type as that of the subject. Inone aspect, the established cell line can be one of a panel of such celllines, wherein the panel can include different cell lines of the sametype of disease and/or different cell lines of different diseasesassociated with expression of the genes of interest. Such a panel ofcell lines can be useful, for example, to practice the present methodwhen only a small number of cells can be obtained from the subject to betreated, thus providing a surrogate sample of the subject's cells, andalso can be useful to include as control samples in practicing thepresent methods.

Once disease and/or skin lesion characterization is established and atreatment protocol is initiated, the methods of the invention may berepeated on a regular basis to monitor the expression profiles of thegenes of interest in the subject. The results obtained from successiveassays may be used to show the efficacy of treatment over a periodranging from several days to months. Accordingly, another aspect of theinvention is directed to methods for monitoring a therapeutic regimenfor treating a subject having skin cancer. A comparison of theexpression profile or mutations in the nucleic acid sequence of thenucleic acid molecule prior to and during therapy will be indicative ofthe efficacy of the therapy. Therefore, one skilled in the art will beable to recognize and adjust the therapeutic approach as needed.

The efficacy of a therapeutic regimen for treating a cancer over timecan be identified by an absence of symptoms or clinical signs of theparticular cancer in a subject at the time of onset of therapy. Insubjects diagnosed as having the particular cancer, the efficacy of amethod of the invention can be evaluated by measuring a lessening in theseverity of the signs or symptoms in the subject or by the occurrence ofa surrogate end-point for the disorder.

In addition, such methods may help identify an individual as having apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

When performed in a high throughput (or ultra-high throughput) format,the methods of the invention can be performed on a solid support (e.g.,a microtiter plate, a silicon wafer, or a glass slide), wherein cellsamples and/or genes of interest are positioned such that each isdelineated from each other (e.g., in wells). Any number of samples orgenes (e.g., 96, 1024, 10,000, 100,000, or more) can be examined inparallel using such a method, depending on the particular support used.Where samples are positioned in an array (i.e., a defined pattern), eachsample in the array can be defined by its position (e.g., using an x-yaxis), thus providing an “address” for each sample. An advantage ofusing an addressable array format is that the method can be automated,in whole or in part, such that cell samples, reagents, genes ofinterest, and the like, can be dispensed to (or removed from) specifiedpositions at desired times, and samples (or aliquots) can be monitored,for example, for expression products and/or mutations in the nucleicacid sequence of the nucleic acid molecules from any one of the geneslisted in Tables 1-8, 10-12, and 15.

Thus, the microarray can be used to monitor the expression level oflarge numbers of genes simultaneously (to produce a transcript image),and to identify genetic variants, mutations and polymorphisms.Polynucleotides used in the microarray may be oligonucleotides that arespecific to a gene or genes of interest in which at least a fragment ofthe sequence is known or that are specific to one or more unidentifiedcDNAs which are common to a particular cell type, developmental ordisease state. In order to produce oligonucleotides to a known sequencefor a microarray, the gene of interest is examined using a computeralgorithm which starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The“pairs” will be identical, except for one nucleotide which preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

According to another aspect of the present invention, a kit is providedthat is useful for detecting cancer in a cell or tissue, e.g., using themethods provided by the present invention for characterizing a skinlesion in a subject. In one embodiment, a kit of the invention includesa skin sample collection device and one or more probes or primers thatselectively bind to one or more of the nucleic acid molecules in any ofTables 1-8, 10-12, and 15. In another embodiment, the kit includes oneor more applicators in addition to or instead of the skin samplecollection device. Such applicators are useful for in situ analysis ofgene expression on the skin of a subject. For example, an applicator maybe used to apply detectably labeled probes for visual detection ofexpressed RNA to characterize the skin lesion.

In another embodiment, a kit of the invention includes a probe thatbinds to a portion of a nucleic acid molecule in any of Tables 1-8,10-12, and 15. In another embodiment, the kit further includes amicroarray that contains at least a fragment of a gene or a nucleic acidmolecule or a protein product of any one of the genes listed in Tables1-8, 10-12, and 15. In some embodiments, many reagents may be providedin a kit of the invention, only some of which should be used together ina particular reaction or procedure. For example, multiple primers may beprovided, only two of which are needed for a particular application.

In another embodiment, the kit of the invention provides acompartmentalized carrier including a first container containing a pairof primers. The primers are typically a forward primer that selectivelybinds upstream of a gene on one strand, and a reverse primer thatselectively binds upstream of a gene on a complementary strand.Optionally the kits of the present invention can further include aninstruction insert, e.g., disclosing methods for sample collection usingthe sample collection device and/or exemplary gene expression profilesfor comparison with the expression profile of the sample taken from thesubject.

The following examples are provided to further illustrate the advantagesand features of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

Example 1 RNA Quantitation and Profiling

The core hypothesis of this study is that epidermal cells overlying insitu or invasive melanoma, including but not limited to the stratumcorneum, stratum lucidum, and stratum granulosum, can be recovered byadhesive means and that the quality and quantity of gene expression inthe form of RNA contained within this sample is differently expressedthan from a nearby epidermal sample, i.e. that the sampled RNA isdiagnostic because of the underlying melanoma. It has been previouslyshown that changes in gene expression of specific genes are detectablein epidermal hyperplasia overlying cutaneous human melanoma samplesobtained from surgical specimens of the epidermis (McCarty et al.,2003).

The present study is divided into two separate phases, a samplecollection and characterization phase (phase 1) and an RNA profilingphase (phase 2). In phase 1 the tape stripped specimens and biopsiedsample collections were performed by the principal investigator ortrained individuals delegated by the principal investigator to obtainthe biopsy sample at various sites. All biopsies are subject to standardhistopathologic analysis. The RNA profiling phase (Phase 2), includes,but is not limited to RNA purification and hybridization to DNAmicroarrays for gene expression profiling.

Materials and Reagents.

Adhesive tape was purchased from Adhesives Research (Glen Rock, Pa.) inbulk rolls. These rolls were custom fabricated into small circulardiscs, 17 millimeters in diameter, by Diagnostic Laminations Engineering(Oceanside, Calif.). Human spleen total RNA was purchased from Ambion(catalogue #7970; Austin, Tex.). RNeasy RNA extraction kit was purchasedfrom Qiagen (Valencia, Calif.). Reverse transcriptase, PCR primers andprobes, and TaqMan Universal Master Mix, which included all buffers andenzymes necessary for the amplification and fluorescent detection ofspecific cDNAs, were purchased from Applied Biosystems (Foster City,Calif.). MELT total nucleic acid isolation system was purchased fromAmbion (Austin, Tex.).

RNA Isolation.

RNA was extracted from tapes using either pressure cycling technology(PCT; Garrett, Tao et al. 2002; Schumacher, Manak et al. 2002) or MELTtotal nucleic acid system. Tapes were extracted in pairs by insertioninto a PULSE™ tube (Pressure Biosciences, Gaithersburg, Md.) with 1.2mls of buffer RLT (supplied in the Qiagen RNeasy kit). PULSE™ tubes wereinserted into the PCT-NEP2017 pressure cycler and the sample wasextracted using the following parameters: room temperature; 5 pressurecycles of 35 Kpsi with pressure held for 20 seconds at the top andbottom of each cycle. After pressure extraction the buffer was removedand used to process the remaining tapes used to strip that site; thebuffer was then processed according to the standard Qiagen RNeasyprotocol for the collection of larger RNAs (>200 nucleotides) byapplication to a purification column to which large RNA molecules (i.e.mRNAs) bind, while the column flow-through is saved for microRNApurification. The column flow-through, which contains miRNA separatedfrom mRNA, is processed according to the Qiagen miRNA purificationprocedure (on the world wide web atqiagen.com/literature/protocols/pdf/RY20.pdf) to purify the microRNA.RNA from the 2 sites stripped on each subject was pooled to create asingle sample from each subject.

RNA Isolation Using MELT Total Nucleic Acid Protocol.

Tapes were extracted in a 2 ml eppendorf tube with 192 ml MELT bufferplus 8 ml of MELT cocktail and vortexed for 10 minutes at roomtemperature. The MELT lysates were transferred to the dispensed bindingbead master mix after spinning down for 3 minutes at >10,000×g andwashed with 300 ml of Wash Solution 1 and 2. RNAs were eluted in 100 mlof elution solution.

Quantitation of mRNA.

Experimental data is reported as the number of PCR cycles required toachieve a threshold fluorescence for a specific cDNA and is described asthe “C_(t)” value (Gibson, Heid et al. 1996; Heid, Stevens et al. 1996;AppliedBiosystems 2001). Quantitation of total RNA mass was performed aspreviously described (Wong, Tran et al. 2004). Briefly, RNA massrecovered from tapes is determined by using quantitative RT-PCR withreference to a standard curve (C_(t, actin) vs. log [RNA];AppliedBiosystems 2001) created from commercially purchased human spleentotal RNA. The average of 6 replicate C_(t, actin) values was used tocalculate the concentration of RNA in a sample with reference to thestandard curve.

RNA Amplification and Array Hybridization.

RNA was isolated by the Multi-Enzymatic Liquefaction of Tissue method(Ambion, Austin, Tex.) and amplified using the WT-Ovation picoamplification system (NuGen, San Carlos, Calif.). The amplified RNA washybridized to Affymetrix U133 plus 2.0 microarray and data wereprocessed and analyzed using R from Bioconductor.

Sample Size.

Sample size calculations are presented in Example 2. This analysispredicts that in order to find 25-40 genes with high predictive value(p<0.001) for discriminating benign nevi from melanoma thenapproximately 30 melanomas and 30 non-melanoma lesions are needed.

Preprocessing GeneChip Data.

The image files from scanning the Affymetrix GeneChips with theAffymetrix series 3000 scanner will be converted using GCOS software(Affymetrix) to “CEL” format files. Normalization of CEL files will becarried out using software from the Bioconductor suite (on the worldwide web at bioconductor.org). In particular, a robust multiarrayanalysis with adjustments for optical noise and binding affinities ofoligonucleotide probes (Wu et al., 2006; and Wu et al., 2004) asimplemented by the function “just.gcrma” in the “gcrma” package will beused to normalize the GeneChip Data.

Statistical Approach for Microarray Data Analysis.

Two generic statistical problems are addressed in this proposal: (i)identifying genes that are differentially expressed in different classesof lesions (i.e. melanoma versus non-melanoma lesions) and (ii) forming(and evaluating) rules for classification of melanoma and non-melanomalesions into groups based on gene expression data.

The most important grouping divides melanoma from non-melanoma on thebasis of biopsy results. The methods that will be used to address theproblems identified above are now standard in the statistical evaluationof microarray data (for reviews see Smyth et al., 2003; and Lee, 2004)).These methods have been applied by others to data from Affymetrix arraysto study gene expression in prostate cancer (Stuart et al., 2004), tocharacterize changes in gene expression subsequent to HIV infection(Mitchell et al., 2003), and to develop a high throughput genotypingplatform (Wolyn et al., 2004; and Borevitz et al., 2003). Foridentifying differentially expressed genes, permutation based estimatesof false discovery rates (reviewed in Efron et al., 2002) are preferred.Scripts for the R quantitative programming environment were developed toimplement these methods in our previous work, but will likely use oradapt the “siggenes” package from the Bioconductor suite in thisproject. The development of classification rules will rely on resamplingmethods (k-fold cross-validation, the 632 plus bootstrap, and/or bagging(Hastie et al., 2001) applied to the naive Bayes classifier and thenearest shrunken centroid classifier (Tibshirani et al., 2002) and thesupport vector machine (SVM) which both performed well in classifyingprostate tissues as malignant or benign, used in our previous work. Theimplementation likely to be used is to perform k-fold cross-validation.Within each of the k train/test cycles an initial screen of the trainingdata for differentially expressed genes is performed and genes areordered according to their posterior probability of differentialexpression. Naive Bayes and nearest shrunken centroid classifiers basedon the r genes with the highest posterior probability of differentialexpression are formed choosing enough values of r between 1 and 1024 toallow accurate interpolation of the classification error rate. The “onese rule” (Brieman et al., 1984) is applied to the error rates for thetest sets to choose the classifier that minimizes the error rate. ForSVM, an internal 632+ bootstrap is applied to each training sample toselect the number of genes to be used in forming the classifier. The “1se rule” error rates from the k test sets are used to characterize theclassification accuracy.

In addition to the use of univariate and multivariate statisticalanalysis tools, sophisticated bioinformatic analysis approaches willhelp make sense of possible biological links between the genes found tobe differentially expressed between, e.g., melanoma and non-melanomasamples. These approaches will focus on the analysis of genetic networksand pathways (Edelman et al., 2006; Kong et al., 2006; and Pang et al.,2006) and have been implemented in software packages such as Ingenuity(on the world wide web at ingenuity.com) and MetaCore (on the world wideweb at genego.com). The identification of the biological links betweengenes that emerge from a gene expression microarray analysis can helpput into context the biological meaningfulness of their expressionpatterns as well as help reduce the set of differentially expressedgenes to be represented on a diagnostic panel based on their biology.The end result of this analysis will be to define a candidate expressionclassifier that will be validated in future, larger clinical trials.

QC Metrics for RNA, Amplified cDNA and Microarray Data.

Following informed consent, the suspicious pigmented lesion was tapestripped using EGIR and then biopsied as per standard of care. Theresulting RNA isolated from the EGIR tape was amplified and profiled onthe Affymetrix U133 plus 2.0 GeneChip. Microarray data were normalizedby the GCRMA algorithm. To assure high quality of microarray data aregenerated, QC metrics were established for RNA, amplified cDNA andmicroarray data. The quality of RNA was assessed by capillaryelectrophoresis using the Experion system (Biorad, Hercule, Calif.) andRNA with at least one visible 18S rRNA was further processed for RNAamplification. The amplified cDNA was quantified by the Nanodrop systemand quality of the amplified cDNA was also assessed by the Experionsystem. The yield of the amplified cDNAs greater than 5 mg and theaverage size distribution of the cDNAs greater than 750 nt were carriedforward for microarray hybridization. Quality of the array data wasfurther assessed using simpleaffy program in R and the array data withscaling factor less than 5.0 and % present call greater than 30% wereused for further data analysis.

Class Modeling—PAM.

After passing the array data QC, 14 melanomas, 40 dysplastic nevi and 12normal skin specimens were further analyzed. First, gene expressionvalues less than 50 across all samples were filtered out and 16716probesets were tested. These 16716 probesets were subjected to astatistical analysis for differentially expressed genes among melanomas,dysplastic nevi and normal skin using ANOVA (p<0.05), multiple testingcorrection algorithm (Westafall and Young permutation) and falsediscover rate (FDR) of 0.05. As indicated above, of the original 117genes, an 89 gene panel (Table 2) was found to be a potential melanomaclassifier. Further testing identified a 5-gene classifier (Table 3), a30-gene classifier (Table 4) that includes newly identified genes, a20-gene classifier (Table 5) that includes newly identified genes, and a19-gene classifier that includes newly identified genes, which may allbe used to discriminate melanomas from atypical nevi. The genes andrespective classifier panels were analyzed using the Prediction Analysisof Microarrays (PAM) software freely available from Stanford University(Stanford, Calif.).

The PAM software uses a modification of the nearest centroid method,which computes a standardized centroid for each class in a training set.This refers to the average gene expression for each gene in each classdivided by the within-class standard deviation for that gene. Nearestcentroid classification takes the gene expression profile of a newsample, and compares it to each of these class centroids. The class,whose centroid it is closest to, in squared distance, is the predictedclass for that new sample.

These genes were all subjected to a hierarchical clustering analysis andthe melanoma specimens grouped together and were clearly distinguishedfrom dysplastic nevi and normal skin. In addition, there are threedistinct classes of dysplastic nevi; one is grouped together with normalskin and the second one was in between normal skin and melanomas, whilethe third one was grouped together with melanomas. These data suggeststratum corneum RNA, harvested by tape stripping with EGIR, can be usedto distinguish melanoma from dysplastic nevi in suspiciously pigmentedlesions.

The analysis of the genes as potential melanoma classifiers todiscriminate between melanomas and dysplastic nevi was performed usingt-test (p<0.01), FDR (0.05) and 2-fold difference between melanomas anddysplastic nevi. Of the original 117 genes, an 89 gene panel (Table 2)was found to be a potential melanoma classifier and functions of these89 genes were subjected to Ingenuity Pathway Analysis (IPA) (Ingenuity,Redwood City, Calif.). Among them, 15 genes are involved hair and skindevelopment and function, 18 genes are involved in cellular development,16 genes are involved in cellular growth and proliferation and 24 genesare related to cancer. Thus, differentially expressed genes are genesrelated to biological functions in melanocytes including melaninbiosynthesis, melanocyte proliferation, differentiation and development.(See FIGS. 5 and 6).

Class Modeling—Random Forests.

Additional work, in which 31 melanomas, 71 atypical nevi, and 15 normalskin controls were analyzed by GeneChip assay, identified 284differentially expressed genes (p<0.001, false discovery rate q<0.05).Hierarchical cluster analysis of these genes showed that melanomas canbe distinguished from atypical nevi and normal skin, and, suggested theexistence of different classes of atypical nevi (FIG. 7). Several of thegenes were found by Ingenuity Pathways analysis to play a role inmelanocyte development and function, as well as, skin development,cellular proliferation, and cancer. These findings further demonstratedthat the presence of melanoma, directly or indirectly, alters the geneexpression profile of stratum corneum. 229 genes were subject to RandomForests analysis and 61 of those 229 genes were found to discriminatemelanoma from atypical nevi (see FIG. 8).

Random Forests analysis is based on Bagging Predictors, which is amethod for generating multiple versions of a predictor and using theseto get an aggregated predictor. The aggregation averages over theversions when predicting a numerical outcome and does a plurality votewhen predicting a class. The multiple versions are formed by makingbootstrap replicates of the learning set and using these as new learningsets. Tests on real and simulated data sets using classification andregression trees and subset selection in linear regression show thatbagging can give substantial gains in accuracy. If perturbing thelearning set can cause significant changes in the predictor constructed,then bagging can improve accuracy.

Class Modeling—TREENET®.

82 additional genes were identified (Table 7). TREENET® software(Salford Systems, San Diego, Calif.) was used to identify a 20-genepanel (Table 8), which may all be used to discriminate melanomas fromatypical nevi (see FIG. 9). An additional 19-gene classifier wasidentified from 7199 differentially expressed genes between melanoma andnevi (Table 6; see also FIGS. 11 and 12). The 19-gene classifier wastested against independent samples and shown to be 100% sensitive and88% specific for detection of melanomas. In addition, results from 10melanomas and 10 nevi indicated that qRT-PCR recapitulated the dataobtained using the GeneChip microarray (FIG. 12 and see raw data inTables 13 and 14).

TREENET® is a data mining tool that is based on boosted decision trees.TREENET® is a model building and function approximation system that alsoserves as an initial data exploration tool. It can extract relationshipsin data and calibrate how predictable the outcomes will be, and canhandle both classification and regression problems.

Example 2 Preliminary Power and Sample Size Studies Nevi vs. PrimaryMelanoma

The following sample size and power calculations are based exclusivelyon the large-scale cDNA study data provided in Haqq et at (2005). Thatdata focused on normal skin (n=3 samples), nevi (n=9), primary melanomas(n=6) and metastatic melanomas (n=19). For purposes of the sample sizecalculations, the focus was on the comparison of nevi to primarymelanomas. Power and sample size assessments were calculated based onthe bootstrap strategy outlined by Page et al. Using the raw dataavailable from the Haqq et at (2005) study, gene expressiondifferences—based on all 14,772 probes used in their cDNA assay—betweennevi and primary melanomas were computed using simple t-tests for eachprobe/gene. Note that multiple probes can be used interrogate individualgenes. In addition, normal skin, nevi, and primary melanoma geneexpression differences were also assessed in a three group analysis ofvariance (ANOVA), with the specific contrast between nevi and primarymelanoma computed from this ANOVA. In the figures that follow, threemain parameters are used to assess power and sample size. Table 9(adapted from Page, et al.) shows the number of genes truly or not trulydifferentially expressed, and provides a simple way of describing theseparameters, which are defined as follows (with the color of the curvescorresponding to each parameter provided in parentheses for FIGS. 1A and2A, although FIGS. 1B and 2B focus exclusively on the EDR as definedbelow.

EDR:

Expected Discovery Rate (from Table 9, D/(B+D)). This reflects theexpected proportion of probes/genes that will be declared significantlydifferentially expressed at the defined threshold (here taken to be, forthe most part, p<0.05) that are, in fact, differentially expressedbetween nevi and primary melanomas.

PTP:

Expected Proportion of probes/genes that are True Positives (Table 9,D/(C+D)). This proportion reflects the number of probes/genes showingexpression differences that are likely to be truly differentialexpressed out of the total number of genes whose expression valuesresult in test statistics less than the threshold (e.g., 0.05).

PTN:

Probability of a True Negative result (Table 9, A/(A+B)). Thisprobability concerns probes/genes that are not significantly differentat the assumed threshold (e.g., 0.05) that are, in fact, notdifferentially expressed between skin and melanoma.

TABLE 9 Parameters of Relevance for Assessing the Power of MicroarrayStudies Not differentially Truly differentially Result based on arrayanalysis expressed expressed Genes not significant A B Genes significantC D These columns represent the number of genes found to satisfy thegiven constraint; A = genes found not to be differentially expressed inan array experiment and that are truly not differentially expressed; B =genes that are differentially expressed but are not found to bedifferentially expressed in the array experiment (false negatives); C =genes that are found to be differentially expressed in the arrayexperiment but are not truly differentially expressed (false positives);D = gene found to be differentially expressed in an array experiment andthat are truly differentially expressed.

Nevi Versus Primary Data.

The sample size analysis considered the number of samples necessary to“discover” or identify a probe or gene or set of probes/genes that coulddifferentiate nevi from primary melanomas based on the probe/geneexpression differences obtained by Haqq et al. (2005). FIG. 1 a providesa plot of the EDR, PTP, and PTN as a function of sample size, assuming athreshold for declaring the significance of a probe/gene expressiondifference between nevi and primary melanoma of p<0.05. Thus, from theplot, it appears that in order to “discover,” or identify, 80% of allgenes that have been interrogated on a chip that exhibit a probe/geneexpression difference producing a test statistic p-value <0.05 that willactually reflect a true probe/gene expression difference, a sample sizeof roughly 20 per nevi and primary melanoma group will be needed. Notethat if all 14,772 probes are considered, one is likely to have14,772×0.05=738 exhibit p-values <0.05 by chance alone, of which1,727×0.80=1,381 will likely reflect true gene expression differences atthat significance (i.e., p-value) level. If one is interested inidentifying a smaller set of genes that have a greater probability ofbeing detected as truly differentially expressed, a more stringentthreshold for statistical significance (e.g., 0.001) can be used. Thiswould generate 14,772×0.001=15 genes with p-values <0.001 by chance ofwhich ˜45% (i.e., 34×0.45=7 would likely be truly differentiallyexpressed at that level; see FIG. 1 b; note curves in FIG. 1 b onlycorrespond to the EDR with different assumed type I error rates).

A sample size analysis that considered the contrast results for nevi vs.primary melanoma in the context of an analysis of variance (ANOVA)comparing normal skin, nevi, and primary melanoma was also pursued. Therationale for this is that there are more differences between normalskin and either nevi or primary melanoma than there are between nevi andprimary melanoma (based on an analysis of the Haqq et al (2005) data),and an analysis that considers normal skin gene expression variation mayhelp reduce the noise in the assessment of nevi vs. primary melanomagene expression differences. FIGS. 2 a and 2 b display the results ofthese analyses and provide similar sample size guidelines to thosereflected in FIGS. 1 a and 1 b.

An analysis focusing exclusively on the posterior true probability (PTP)was also considered since, as discussed, there may be many probes/genesthat exhibit differences between nevi and primary melanomas at a certainprobability level purely by chance (given the large number ofprobes/genes interrogated). Thus, the likely fraction of theseprobes/genes that are truly differentially expressed is important toassess. FIGS. 3 a and 3 b reflect the results for different assumedsignificance levels.

Thus, an argument can be made that a study with approximately 20 samplesper nevi and primary melanoma groups would have sufficient power todetect 80% of all genes that are likely to exhibit differentialexpression at a p-value level of 0.05 because they are, in fact,differentially expressed at this level. However, the number of genes (orprobes) contributing to this set of differentially expressed genes islikely to number in the hundreds, if 10,000-30,000 probes are used or5,000-10,000 genes are studied. If interest is in identifying a smallernumber of probes or genes (˜25-40) that have a greater probability ofbeing differentially expressed, say, at a p-value of 0.001, then ˜30nevi and 30 primary melanoma samples would be needed (see FIGS. 1, 2,and 3).

Example 3 Tape Stripping to Recover Nucleic Acids from Normal Skin

The following procedure was used to recover nucleic acids from normalskin (e.g., the mastoid or upper back areas) of a subject.

Tapes were handled with gloved hands at all times. Locate a particularsite that is relatively blemish-free and healthy, unless otherwisespecified by the protocol. Preferred normal skin sites are the mastoidprocess (the bony process behind the ear at the base of the skull) andthe upper back, immediately superior to the scapular spine. Shave thesite if necessary to remove non-vellus hairs. Cleanse the site with analcohol wipe (70% isopropyl alcohol). Let the site air dry completelybefore application of the tape. It is recommended to wait approximately2 minutes to ensure the site is completely dry before application of thetape.

Apply the tape to the skin site. If more than one tape is used, applytapes in sequential order starting from the left side. Use a surgicalskin marker and/or a water soluble marker to mark the location of thetape on the skin in order to align subsequent tapes.

Start the tape harvesting procedure by applying pressure (press on thetape firmly). Ensure that the skin is held taut to ensure that the tapedoes not move while applying pressure. Then remove the tape slowly inone direction. Place the edge of the tape onto the strip at the top ofthe packet with the adhesive surface of the tape facing down to protectthe sample. Put a second tape on the same site; apply pressure firmly asabove. Remove the tape slowly in an opposite direction to that used inthe immediately previous application.

Continue tape stripping by putting additional tapes on the same site,following the steps provided above. The site may stripped with a totalof at least four tapes, unless otherwise specified in the protocol.Place the strip into a storage bag and immediately place the samples ondry ice or into storage at −20° C. or below until analysis.

Example 4 Tape Stripping to Recover Nucleic Acids from Pigmented Lesions

The following procedure was used to recover nucleic acids from pigmentedlesions and/or skin suspected of melanoma of a subject. In contrast tonormal skin, lesional skin should have a preoperative biopsy diameter ofgreater than or equal to about 6 mm, but less than that of the tapedisc. Multiple lesions must be at least about 4 mm apart. The area oftape that touches the lesion should be generously demarcated on the tapewith an insoluble ink pen so that this area may be cut away from thesurrounding tape at the laboratory as part of the RNA extractionprocedure.

As above, tapes were handled with gloved hands at all times. Shave thesite if necessary to remove non-vellus hairs. Cleanse the site with analcohol wipe (70% isopropyl alcohol). Let the site air dry completelybefore application of the tape. It is recommended to approximately 2minutes to ensure the site is completely dry before application of thetape.

Apply the tape to the skin site. If more than one tape is used, applytapes in sequential order starting from the left side. Use a surgicalskin marker and/or a water soluble marker to mark the location of thetape on the skin in order to align subsequent tapes. Apply the tape tothe suspect lesion, which should have a diameter that is greater than orequal to about 6 mm.

Start the tape harvesting procedure by applying pressure directly overthe lesion and avoiding surrounding normal skin (press on the tapefirmly). Ensure that the skin is held taut to ensure that the tape doesnot move while applying pressure. Using a marking pen, demarcate a zonearound the lesion such that the area of the lesion is encompassed withinthe inked boundary and the boundary is approximately 1 mm from thelesion border.

Remove the tape slowly in one direction. Place the edge of the tape ontothe adhesive strip with cells facing down to protect the sample. Put asecond tape on the same site following directions provided above. Repeatuntil the lesion has been stripped a total of at least four times,unless otherwise specified in the protocol. Place the strip into astorage bag and immediately place the samples on dry ice or into storageat −20° C. or below until analysis.

Example 5 Gene Expression Profile to Distinguish Melanoma from AtypicalNevi

The purpose of this study is to determine whether stratum corneum RNA,harvested by tape stripping with EGIR can be used to distinguishmelanoma from atypical nevi in suspicious pigmented lesions. See FIG.4A.

Suspicious pigmented lesions were tape stripped four times using EGIRand then biopsied as per standard of care. Normal, uninvolved skin wastape stripped to serve as a negative control. All biopsies underwentprimary and central review for histopathology. Total RNA was isolatedfrom the tapes using MELT (Ambion, Inc.) and assessed for quality byExperion (Bio-Rad, Inc.) analysis. The yield of RNA was approximately 1ng, as determined by quantitative RT-PCR of the specimen for β-actingene expression. Total RNA (200-500 pg) was then amplified using theWT-Ovation Pico RNA Amplification System (NuGen, Inc.) and assayed forgene expression profile using the U133 plus 2.0 GeneChip (Affymetrix,Inc.).

The resulting RNA isolated from the EGIR tape is then amplified andprofiled on the Affymetrix U133 plus 2.0 GeneChip. Microarray data isnormalized by the GCRMA algorithm. Further analyses by means of ANOVAanalysis (p<0.05) with a false discovery rate of 0.05 and multiplecorrection testing using Westfall and Young permutation identifiedapproximately 117 genes as differentially expressed between melanoma,dysplastic nevi and normal skin (Table 1). Hierarchical clustering ofthese genes showed that the melanoma specimens grouped together and wereclearly distinguished from dysplastic nevi and normal skin (FIG. 4B). Inaddition, 89 of the 117 genes shown in Table 1 were further identified(Table 2) as potential discriminators between melanoma and dysplasticnevi (p<0.01, false discovery rate q<0.05). When these 89 genes weresubjected to Ingenuity Pathways analysis many were found to play rolesin melanoma, hair and skin development and function, cellulardevelopment, cellular growth and proliferation and cancer. Thesefindings demonstrate that EGIR-harvested RNA from suspicious pigmentedskin lesions can be used to differentiate melanoma from dysplastic nevi(FIG. 4C). Further, these results suggest that the gene expressionprofile of stratum corneum is altered, either directly or indirectly, bythe presence of melanoma (FIG. 4D).

In subsequent studies that compared normal and inflamed skin, sequentialapplication of four small tapes at the same skin site recovered enoughintact RNA to perform quantitative reverse-transcription polymerasechain reaction (qPCR) assay and DNA microarray analysis forinvestigation of gene expression. The latter assay was performed usingthe Affymetrix HG-U133A GeneChip following two rounds of amplificationof 10 ng of total RNA sample that produced 30-80 μg of anti-sense RNA.Comparison of results from two subjects, each sampled at three separatesites, showed 12% intra- and inter-subject variance in genemeasurements, a result that is well within the Affymetrix specifiedcoefficient of variation (CV) for GeneChip assay. Of note is thatdifferential expression of Y-chromosome genes was observed, a resultthat accurately distinguished the different genders of the 2 subjects.GeneChip assay was then performed on RNA isolated from tape strippingeach of 3 subjects from normal, water occluded, and sodium laurylsulfate-irritated study groups. The majority of 100 genes, whoseexpression is most significantly altered between untreated andSLS-treated skin showed, were already known to be involved in tissueinflammation and injury functions. Thus, RNA harvested by EGIRtechnology is more than adequate for microarray-based gene expressionprofiling and appropriately reflects the pathologic state of skin.

Recent work by Benson et at (2006) demonstrates that RNA can berecovered from psoriatic lesions and that the general RNA expressionprofile of tape strip recovered RNA is consistent with biopsy RNAderived from lesions on the same patient. Further work (see U.S. Pat.No. 7,183,057, incorporated herein by reference) has shown thatpsoriatic lesions can be sampled with tape during treatment with Enbreland that strong correlations could be made between gene expression inweek one of treatment and clinical response at weeks 4 and 8. This workfurther establishes the credentials of tape stripping for the recoveryof physiologically relevant RNA from the surface of the skin.

Example 6 Gene Expression Profile to Distinguish Solar Lintigenes fromMelanoma, Atypical Nevi, and/or Normal Skin

The purpose of this study is to determine whether stratum corneum RNA,can be used to distinguish solar lentigines from melanoma, atypicalnevi, and/or normal skin in suspicious pigmented lesions.

Suspicious pigmented lesions were tape stripped as above and thenbiopsied as per standard of care. Normal, uninvolved skin was tapestripped to serve as a negative control. All biopsies underwent primaryand central review for histopathology. Total RNA was isolated providedabove and then amplified and profiled, as provided above. 1600 genesthat were differentially expressed among solar lentigines and normalskin controls were selected. Further testing identified a 103-geneclassifier (Table 10), which may be used to discriminate solarlentigines from normal pigmented skin (FIGS. 14 to 16).

Additional work, in which 11 solar lentigo samples, 12 atypical nevisamples, and 8 basal cell carcinoma (BCC) samples were analyzed usingANOVA (p<0.05), FDR (p<0.05) and multiple test correction to identify 82differentially expressed genes (Table 11). Heirarchical analysis of the82-gene classifier shows that it may be used to discriminate betweensolar lentigines and atypical nevi and/or basal cell carcinoma (BCC)(FIG. 17). Finally, a 32-gene classifier (Table 12) was identified,which may be used to discriminate between solar lentigines and lentigomaligna (FIG. 18). The genes and respective classifier panels wereanalyzed using the Prediction Analysis of Microarrays (PAM) softwarefreely available from Stanford University (Stanford, Calif.).

An additional 28-gene classifier was identified from 2437 differentiallyexpressed genes between lentigo maligna and solar lentigo was identifiedby TREENET® analysis (Table 15; see also FIG. 19). In addition, resultsfrom 26 lentigo maligna and 34 solar lentigo samples indicated thatqRT-PCR recaptilated data obtained using the GeneChip microarray (seeraw data in Tables 16-21).

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TABLE 1 Entrez Entrez Gene ID Gene ID Entrez for for Gene ID namematched term synonym description Human Mouse for Rat ACTR1B 202135_s_at2310066K23Rik, ARP1 actin-related 10120 226977 (includes AA960180,protein 1 homolog B, EG: 10120) ACTR1B, centractin beta (yeast)AI851923, ARP1B, CTRN2, MGC36526 ANGEL1 213099_at 1110030H02Rik, angelhomolog 1 23357 68737 362765 KIAA0759, (Drosophila) mKIAA0759,RGD1306238 ANKRD13B 227720_at AW124583, ankyrin repeat domain 124930268445 360575 B930093C12Rik, 13B FLJ20418, FLJ25555, RGD1564005 ANKRD44228471_at 4930444A19Rik, ankyrin repeat domain 44 91526 329154 301415A130096K20, E130014H08Rik, LOC91526, MGC21968, MGC70444, RGD1561893ARHGEF19 226857_at 6030432F23, Rho guanine nucleotide 128272 213649362648 6430573B13Rik, exchange factor (GEF) 19 FLJ33962, RP4- 733M16.1,WGEF ATPBD4 238662_at 5730421E18Rik, ATP binding domain 4 89978 66632362191 MGC14798, RGD1310006 BARX2 210419_at 2310006E12Rik, BarH-likehomeobox 2 8538 12023 Barx2b, MGC133368, MGC133369 BDNF 206382_s_atMGC105254, brain-derived 627 12064 24225 MGC34632 neurotrophic factorBLOC1S1 202592_at AI839753, biogenesis of lysosome- 2647 14533 288785BLOC-1 subunit related organelles 1, BLOS1, complex-1, subunit 1GCN5-like protein 1, GCN5L1, MGC87455, RT14 BTG2 201236_s_at AA959598,Agl, BTG family, member 2 7832 12227 29619 An, an-1, APRO1, MGC126063,MGC126064, PC3, TIS21 C16ORF48 223407_at AI606951, chromosome 16 open84080 102124 291975 DAKV6410, reading frame 48 DKFZP434A1319,E130303B06Rik, RGD1307357 C6ORF218 244829_at MGC40222 chromosome 6 open221718 reading frame 218 C8ORF13 233641_s_at A030013D21, chromosome 8open 83648 219148 498533 BC065085, reading frame 13 D8S265,DKFZp761G151, MGC120649, MGC120650, MGC120651, RGD1561302 CCDC95227286_at AI225782, coiled-coil domain 283899 233875 AI854876,containing 95 Ccdc85, FLJ00079, FLJ90652, MGC31515 CCHCR1 37425_g_atC6orf18, HCR, coiled-coil alpha-helical 54535 240084 406196 MGC126371,rod protein 1 MGC126372, MGC28303, RGD: 1302992, SBP CIRBP 230142_s_atA18 HNRNP, cold inducible RNA 1153 12696 81825 CIRP, R74941 bindingprotein CLSTN2 219414_at 2900042C18Rik, calsyntenin 2 64084 64085 171394AI448973, alcagamma, CS2, Cst-2, CSTN2, FLJ39113, FLJ39499, KIAA4134,MGC119560, mKIAA4134 COL7A1 217312_s_at AW209154, collagen, type VII,alpha 1294 12836 301012 EBD1, EBDCT, 1 (epidermolysis bullosa, EBR1dystrophic, dominant and recessive) DACH1 205471_s_at, AI182278, Dac,dachshund homolog 1 1602 13134 205472_s_at, DACH, (Drosophila) 228915_atE130112M23Rik, FLJ10138 DCT 205337_at, DT, dopachrome tautomerase 163813190 290484 205338_s_at RGD1564975, (dopachrome delta- slaty, slt,TRP-2, isomerase, tyrosine-related TYRP2 protein 2) DOCK10 219279_at9330153B10RIK, dedicator of cytokinesis 55619 210293 301556A630054M16Rik, 10 DKFZp781A1532, DRIP2, Jr4, Jr5, mKIAA0694, Nbla10300,R75174, RGD1561963, ZIZ3, Zizimin3 DRAP1 1556181_at 2310074H19Rik,DR1-associated protein 1 10589 66556 293674 MGC156767, (negativecofactor 2 alpha) NC2-ALPHA, negative cofactor 2 alpha EDNRB204271_s_at, ABCDS, endothelin receptor type B 1910 13618 50672206701_x_at AU022549, Ednra, ET&gt; B&lt;, ET-B, ETB RECEPTOR, ETBR,ETRB, GUSB, HSCR, HSCR2, Sox10m1 EFNA4 205107_s_at EFL-4, EPHRINephrin-A4 1945 13639 310643 A4, Epl4, EPLG4, LERK-4, MGC125826 EHD245297_at BC027084, EH-domain containing 2 30846 259300 361512C130052H20Rik, MGC25606, MGC38650, MGEPS, PAST2 ETS1 224833_at AI196000,v-ets erythroblastosis 2113 23871 24356 AI448617, C- virus E26 oncogeneETS1, homolog 1 (avian) D230050P06, Etsoncb, EWSR2, FLJ10768, MGC124638,MGC130355, MGC18571, p42 ETS1, p51 ETS1, Tpl1 FAM33A 225684_at1110001A07Rik, family with sequence 348235 66140|625534 287598 C78640,similarity 33, member A EG625534, FLJ12758, MGC109093, MGC110975,MGC151378, RGD1307084 FGFR1 210973_s_at, AW208770, fibroblast growthfactor 2260 14182 79114 211535_s_at BFGFR, C-FGR, receptor 1(fms-related CD331, CEK, tyrosine kinase 2, Pfeiffer FGF1 syndrome)RECEPTOR, FGFBR, FGFR1- IIIC, Fgfr1c, FLG, Flk2, FLT2, H5, HBGFR, KAL2,N-SAM FOXO1A 202723_s_at Afxh, AI876417, forkhead box O1A 2308 5645884482 FKH1, FKHR, (rhabdomyosarcoma) FKHR1, Forkhead, FOXO1 FOXP1223936_s_at 12CC4, forkhead box P1 27086 108655 297480 3110052D19Rik,4932443N09Rik, AI461938, AW494214, FLJ23741, hFKH1B, HSPC215, MGC116362,MGC12942, MGC88572, MGC99551, QRF1 FRAT2 209864_at MGC10562, frequentlyrearranged in 23401 212398 MGC37615 advanced T-cell lymphomas 2 GCLM203925_at Gamma gclm, glutamate-cysteine ligase, 2730 14630 29739 Gammamodifier subunit glutamylcysteine synthase (regulatory), GAMMAGLUTAMYLCYSTEINE SYNTHETASE, Gcs Ls, Gcs, Regulatory, GCS- L, GCS1,Gcslc, GLCLR, glutamat-cystein ligase, regulatory subunit GGA3209411_s_at C230037M19Rik, golgi associated, gamma 23163 260302 360658KIAA0154, adaptin ear containing, mKIAA0154 ARF binding protein 3 GLUL200648_s_at GLNS, glutamate-ammonia 2752 14645 Glutamine ligase(glutamine Synthase, synthetase) GLUTAMINE SYNTHETASE, GS, MGC128403,PIG43 GPR161 214104_at FLJ33952, G- G protein-coupled 23432 240888289180 protein coupled receptor 161 receptor af091890, Gm208, Gm208Gpr,RE2, RGD1563245 HEY2 219743_at CHF1, GRL, hairy/enhancer-of-split 2349315214 155430 HERP1, HESR2, related with YRPW motif 2 HRT2, MGC10720HIST2H2AA3 214290_s_at AI448581, H2A, histone cluster 2, H2aa3 833715267 365877 H2a-615, H2A.2, H2A/O, H2A/q, H2AFO, Hist2, HIST2H2AA,Hist2h2aa1 ID1 208937_s_at AI323524, inhibitor of DNA binding 3397 1590125261 D2Wsu140e, ID, 1, dominant negative ID-1H, ID125A,helix-loop-helix protein Idb1, MGC156482 KALRN 227750_at 2210407G14Rik,kalirin, RhoGEF kinase 8997 545156 84009 AV235988, DUET, Duo,E530005C20Rik, FLJ16443, Gm539, HAPIP, KALIRIN, Kalirin7, Pcip10, TRADKDELR1 200922_at 8030486F04Rik, KDEL (Lys-Asp-Glu- 10945 68137 361577AW215843, Leu) endoplasmic ERD2, ERD2.1, reticulum protein retentionHDEL, KDEL receptor 1 RECEPTOR, Kdelr, MGC109169, PM23 KIAA0738210529_s_at 2810407D09Rik, KIAA0738 gene product 9747 77574 3623533321401G04Rik, A230020K05Rik, AI848529, RGD1565474 KIT 205051_s_at Bs,C-KIT, c-Kit v-kit Hardy-Zuckerman 4 3815 16590 64030 Gnnk+, CD117,feline sarcoma viral Fdc, SCFR, Ssm, oncogene homolog Tr Kit, white-spotted LGR4 230674_at 9130225G07, leucine-rich repeat- 55366 107515286994 A930009A08Rik, containing G protein- GPCR48, GPR48 coupledreceptor 4 LHX2 211219_s_at ap, apterous, LIM homeobox 2 9355 16870296706 (includes hLhx2, Lh-2, EG: 9355) LH2A, Lhx2, Lim2, MGC138390 LMO4209204_at A730077C12Rik, LIM domain only 4 8543 16911 362051 Crp3,Etohi4, MGC105593 LOC254100 1557131_at hypothetical protein 254100LOC254100 LRIG1 236173_s_at, D6Bwg0781e, leucine-rich repeats and 2601816206 312574 238339_x_at DKFZP586O1624, immunoglobulin-like Img, LIG-1domains 1 MED28 222635_s_at 1500003D12Rik, mediator of RNA 80306 66999305391 AI451633, polymerase II AU045690, transcription, subunit 28DKFZP434N185, homolog (S. cerevisiae) EG1, FKSG20, magicin, RGD1305875MKL1 215292_s_at AI852829, megakaryoblastic 57591 223701 315151 AMKL,leukemia (translocation) 1 AW743281, AW821984, BSAC, MAL, MRTF-A MLANA206426_at, A930034P04Rik, melan-A 2315 77836 293890 206427_s_at MART-1,MELAN-A, MGC130556 MLLT6 225628_s_at AF17, myeloid/lymphoid or 4302246198 303504 AI315037, mixed-lineage leukemia FLJ23480 (trithoraxhomolog, Drosophila); translocated to, 6 MLPH 218211_s_at 2210418F23Rik,melanophilin 79083 171531 316620 5031433I09Rik, AW228792, D1Wsu84e,l(1)- 3Rk, 11Rk3, ln, MGC2771, MGC59733, SLAC2-A MYEF2 222771_s_at,9430071B01, myelin expression factor 2 50804 17876 362207 232676_x_atFLJ11213, HsT18564, KIAA1341, MEF-2, MGC109392, MGC87325, mKIAA1341,MST156, MSTP156 MYL6B 204173_at 5730437E04Rik, myosin, light chain 6B,140465 216459 317454 Atrial Myosin alkali, smooth muscle and Light Chain1, non-muscle BC037527, MGC41229, MLC1SA, RGD1560334 MYO5A 227761_at9630007J19Rik, myosin VA (heavy chain 4644 17918 25017 AI413174, 12,myoxin) AI661011, Br Myosin5a, d- 120J, Dbv, Dop, flail, flr, GS1,hcBM-V, MVa, MYH12, MYO5, myosin V, MYOSIN VA, MYOSIN VA EXONCONTAINING, MYOVA, MYOXIN, MYR12, Sev-1 NBL1 37005_at D1S1733E,neuroblastoma, 4681 17965 50594 D4H1S1733E, suppression of DAN, Dana,tumorigenicity 1 DAND1, MGC123430, MGC8972, NB, NO3 NFIB 230791_at6720429L07Rik, nuclear factor I/B 4781 18028 29227 CTF/NF1B,E030026I10Rik, NF1-B, NFI- RED, NFIB2, NFIB3, Nuclear factor 1/B OSTM1218196_at 1200002H13Rik, osteopetrosis associated 28962 14628 445370AW123348, transmembrane protein 1 GIPN, GL, HSPC019 PDK3 221957_at2610001M10Rik, pyruvate dehydrogenase 5165 236900 296849 AI035637,kinase, isozyme 3 MGC6383 PKD1 241090_at FLJ00285, polycystic kidneydisease 5310 18763 24650 mFLJ00285, 1 (autosomal dominant) MGC118471,PBP, PC-1, POLYCYSTIN1 PLEKHA5 220952_s_at 2810431N21Rik, pleckstrinhomology 54477 109135 246237 AI428202, domain containing, familyAK129423, A member 5 Ayu21-9, FLJ10667, FLJ31492, Gt(pU21)9Imeg, Image:3710928, KIAA1686, MGC38455, PEPP2, TRS1 PLP1 210198_s_at DM20, jimpy,jp, proteolipid protein 1 5354 18823 24943 MMPL, Msd,(Pelizaeus-Merzbacher PLP, PLP/DM20, disease, spastic paraplegia PMD, 2,uncomplicated) PROTEOLIPID, RSH, SPG2 PLXNC1 213241_at 2510048K12Rik,plexin C1 10154 54712 362873 AW742158, CD232, Plexin C1, VESPR PPP3CA202425_x_at 2900074D19Rik, protein phosphatase 3 5530 19055 24674AI841391, (formerly 2B), catalytic AW413465, subunit, alpha isoformCalcineurin, (calcineurin A alpha) Calcineurin A Alpha, CALN, CALNA,CALNA1, CCN1, CN, CnA, CnA- alpha, CNA1, MGC106804, Pp2b Subunit A,PPP2B PRKCSH 200707_at 80K-H, AGE- protein kinase C substrate 5589 19089300445 R2, G19P1, 80K-H PCLD, PLD, PLD1 PRKD3 222565_s_at 4930557O20Rik,protein kinase D3 23683 75292 313834 5730497N19Rik, EPK2, MGC47171,nPKC-NU, PKC- NU, PKD3, PRKCN PRMT1 206445_s_at 6720434D09Rik, proteinarginine 3276 15469 60421 ANM1, methyltransferase 1 AW214366, HCP1,heterogeneous ribonucleooproteins methyltransferase- like 2, Hnmt112,Hramt, HRMT1L2, IR1B4, Mrmt1 PSCD3 225147_at AI648983, pleckstrinhomology, 9265 19159 116693 ARNO3, Sec7 and coiled-coil CYTOHESIN-3,domains 3 GRP1, KIAA4241, MGC124579, mKIAA4241, Sec7, Sec7C PTPRF200635_s_at, AA591035, protein tyrosine 5792 19268 360406 200637_s_atFLJ43335, phosphatase, receptor FLJ45062, type, F FLJ45567, LAR, Larptp2b, LARFN5C, LARS PTPRM 1555579_s_at HR-PTPU, protein tyrosine 579719274 29616 KIAA4044, phosphatase, receptor MGC90724, type, M mKIAA4044,PTP-MU, PTPRL1, R-PTP- MU, RPTPM, RPTPU PVRL1 225211_at AI835281,poliovirus receptor- 5818 58235 192183 AW549174, related 1 (herpesvirusCD111, entry mediator C; nectin) CLPED1, ED4, HIgR, HVEC, MGC142031,MGC16207, NECTIN-1, Nectin1 alpha, Nectin1 delta, OFC7, PRR, PRR1, PVRR,PVRR1, SK-12 RAB40C 227269_s_at RAB40, RAR3, RAB40C, member RAS 57799224624 359728 RARL, RASL8C oncogene family RASSF3 230466_s_at AW212023,Ras association 283349 192678 362886 AW322379, (RalGDS/AF-6) domainMGC119194, family 3 MGC119195, MGC119197, RASSF5 RHOQ 212120_at ARHQ,ras homolog gene family, 23433 104215 85428 RASL7A, Rhot, member Q TC10,TC10 BETA, TC10A SAT1 203455_s_at, AA617398, Ab2- spermidine/spermineN1- 6303 20229 302642 210592_s_at, 402, DC21, acetyltransferase 1213988_s_at, KFSD, 230333_at MGC72945, SAT, Spermidine/spermineN1-acetyl transferase, SSAT, SSAT-1 SDCBP 200958_s_at MDA-9, ST1,syndecan binding protein 6386 53378 83841 SYCL, (syntenin) SYNTENIN,Syntenin-1, TACIP18 SEC61A1 217716_s_at, AA408394, Sec61 alpha 1 subunit29927 53421 80843 222385_x_at AA410007, (S. cerevisiae) HSEC61,rSEC61alpha p, SEC61, Sec61 alpha, SEC61 ALPHA1, SEC61A SEMA3C 236947_at1110036B02Rik, sema domain, 10512 20348 296787 SEMAE, immunoglobulindomain SEMAPHORINE, (Ig), short basic domain, SemE secreted,(semaphorin) 3C SERGEF 220482_s_at, DELGEF, Gef, secretion regulating26297 27414 365243 232983_s_at Gnef, Gnefr, guanine nucleotideMGC141208, exchange factor MGC141209, RGD1563497 SILV 209848_s_atD10H12S53E, silver homolog (mouse) 6490 20431 362818 D12S53E, D12S53Eh,GP100, gp87, ME20, PMEL17, SI, SIL SLC2A4RG 227362_at GEF, HDBP1, SLC2A4regulator 56731 Si-1-2, Si-1-2-19 SLC7A1 212295_s_at 4831426K01Rik,solute carrier family 7 6541 11987 25648 AI447493, (cationic amino acidATRC1, CAT-1, transporter, y+ system), EcoR, ER, ERR, member 1 HCAT1,mCAT- 1, Rec-1, REC1L, REV-1 SRGAP2 1568957_x_at 9930124L22Rik,SLIT-ROBO Rho 23380 14270 360840 AI448945, FBP2, GTPase activatingprotein 2 FNBP2, KIAA0456, RGD1566016, srGAP3 SSBP3 217991_x_at,2610021L12Rik, single stranded DNA 23648 72475 84354 223635_s_at2610200M23Rik, binding protein 3 5730488C10Rik, AI854733, AW551939,CSDP, FLJ10355, LAST, MGC124589, SSDP, SSDP1, Ssdp3 STAM 203544_s_atDKFZp686J2352, signal transducing 8027 20844 498798 (includesRGD1564499, adaptor molecule (SH3 EG: 8027) Stam, STAM1 domain and ITAMmotif) 1 SYNGR2 201079_at CELLUGYRIN, synaptogyrin 2 9144 20973 89815Clast2, MGC102914 TCF7L2 212759_s_at mTcf-4B, mTcf- transcription factor7-like 6934 21416 (includes 4E, TCF-4, 2 (T-cell specific, HMG- EG:6934) TCF4B, TCF4E, box) Tcf7l2 TIMM17A 215171_s_at 17 kDa, translocaseof inner 10440 21854 54311 Mitochondrial mitochondrial membrane importinner 17 homolog A (yeast) membrane translocase, Mitochondrial proteinimport protein 2, mTim17a, TIM17, TIM17A, Timm17 TP53 201746_at bbl,bfy, bhy, tumor protein p53 (Li- 7157 22059 24842 Delta N p53, Fraumenisyndrome) LFS1, MGC112612, P53, TRP53 TP53INP1 235602_at 2700057G22Rik,tumor protein p53 94241 60599 297822 DKFZP434M1317, inducible nuclearprotein 1 FLJ22139, p53DINP1, SIP, SIP18, SIP27, Stinp, Teap, ThymusExpressed Acidic Protein, TP53DINP1, TP53DINP1alpha, TP53INP1A,TP53INP1B, Trp53inp1 TRIB2 202478_at AW319517, tribbles homolog 2 28951217410 313974 C5fw, GS3955, (Drosophila) RGD1564451, TRB-2 TRPM1237070_at 4732499L03Rik, transient receptor 4308 17364 (includesAI606771, potential cation channel, EG: 4308) LTRPC1, subfamily M,member 1 melastatin, MLSN, MLSN1, Trpm1 TSPAN6 209108_at 6720473L21Rik,tetraspanin 6 7105 56496 302313 AI316786, MGC117923, T245, Tm4sf, TM4SF6TSTA3 36936_at AI256181, FX, tissue specific 7264 22122 300036 FXprotein, transplantation antigen MGC113801, P35B P35B, Tstap35b TTC3208073_x_at, 2610202A04Rik, tetratricopeptide repeat 7267 22129 360702210645_s_at AA409221, domain 3 D16Ium21, D16Ium21e, DCRR1,DKFZp686M0150, KIAA4119, mKIAA4119, Mtprd, RNF105, TPRD, TPRDIII TUBB4212664_at AI325297, Beta tubulin, beta 4 10382 22153 29213 tubulin, BETATUBULIN 4 ALPHA, Beta tubulin class iv, beta-5, Beta4 Tubulin, M(beta)4,Tubb, TUBB5, TUBULIN BETA (5-BETA), TUBULIN BETA5 TYR 206630_at albino,Dopa tyrosinase 7299 22173 308800 oxidase, (oculocutaneous albinismMelanogenesis IA) Related Tyrosinase, OCA1A, OCAIA, skc35, Tyr&lt; c-em&gt;, TYROSINASE TYRP1 205694_at b-PROTEIN, tyrosinase-related protein1 7306 22178 298182 brown, CAS2, CATB, GP75, isa, MELANOMA ANTIGEN GP75,TRP, TRP-1, TYRP VDR 204255_s_at NR1I1, VD3R, vitamin D (1,25- 742122337 24873 VITAMIN D dihydroxyvitamin D3) RECEPTOR receptor VGLL4214004_s_at BC048841, vestigial like 4 9686 232334 297523 KIAA0121,(Drosophila) MGC109514, MGC54805, VGL-4 YIPF5 224949_at 2610311I19Rik,Yip1 domain family, 81555 67180 361315 AA408236, Ac2- member 5 256,DKFZp313L2216, FinGER5, SB140, SMAP-5, YIP1A ZFHX1B 1557797_a_at,9130203F04Rik, zinc finger homeobox 1b 9839 24136 311071 203603_s_atD130016B08Rik, KIAA0569, mKIAA0569, SIP-1, SMADIP1, ZEB2, Zfx1b, Zfxh1b1558019_at —: Homo sapiens, clone IMAGE: 4732650, mRNA 233551_atLOC642776: hypothetical protein LOC642776 208646_at RPS14: ribosomalprotein S14 /// similar to ribosomal protein S14 208929_x_at RPL13:ribosomal protein L13 214351_x_at RPL13: ribosomal protein L13 ///similar to ribosomal protein L13 200817_x_at RPS10: ribosomal proteinS10 213296_at —: Transcribed locus 213692_s_at —: — 227957_at —: —232462_s_at FLJ23569:BC040926 227722_at RPS23: ribosomal protein S23217466_x_at RPS2: ribosomal protein S2 /// similar to ribosomal proteinS2 235534_at —: Homo sapiens, clone IMAGE: 5723825, mRNA 230741_at —:Full length insert cDNA clone YX74D05 229067_at LOC653464: Similar toSLIT- ROBO Rho GTPase- activating protein 2 (srGAP2) (Formin0bindingprotein 2)

TABLE 2 name matched term ANKRD44 228471_at ARHGEF19 226857_at ATPBD4238662_at BARX2 210419_at BDNF 206382_s_at BLOC1S1 202592_at C16ORF48223407_at C6ORF218 244829_at C8ORF13 233641_s_at CCHCR1 37425_g_at CIRBP230142_s_at CLSTN2 219414_at COL7A1 217312_s_at DACH1 205472_s_at,228915_at DCT 205337_at, 205338_s_at DOCK10 219279_at DRAP1 1556181_atEDNRB 204271_s_at, 206701_x_at EFNA4 205107_s_at EHD2 45297_at ETS1224833_at FAM33A 225684_at FGFR1 210973_s_at, 211535_s_at FOXO1A202723_s_at GGA3 209411_s_at GPR161 214104_at HIST2H2AA3 214290_s_at ID1208937_s_at KDELR1 200922_at KIAA0738 210529_s_at KIT 205051_s_at LGR4230674_at LHX2 (includes EG: 9355) 211219_s_at LMO4 209204_at LOC2541001557131_at LRIG1 238339_x_at MED28 222635_s_at MKL1 215292_s_at MLANA206426_at, 206427_s_at MLPH 218211_s_at MYEF2 222771_s_at, 232676_x_atMYO5A 227761_at NBL1 37005_at OSTM1 218196_at PDK3 221957_at PKD1241090_at PLEKHA5 220952_s_at PLP1 210198_s_at PLXNC1 213241_at PRKCSH200707_at PRKD3 222565_s_at PRMT1 206445_s_at PSCD3 225147_at PTPRF200637_s_at PTPRM 1555579_s_at RAB40C 227269_s_at RASSF3 230466_s_atRHOQ 212120_at RPL13 214351_x_at RPS23 227722_at SAT1 203455_s_at,213988_s_at, 230333_at SDCBP 200958_s_at SEC61A1 222385_x_at SEMA3C236947_at SERGEF 232983_s_at SILV 209848_s_at SLC2A4RG 227362_at SLC7A1212295_s_at SSBP3 217991_x_at, 223635_s_at STAM (includes EG: 8027)203544_s_at SYNGR2 201079_at TCF7L2 (includes EG: 6934) 212759_s_atTIMM17A 215171_s_at TRIB2 202478_at TRPM1 (includes EG: 4308) 237070_atTSPAN6 209108_at TTC3 208073_x_at, 210645_s_at TUBB4 212664_at TYR206630_at VDR 204255_s_at YIPF5 224949_at ZFHX1B 1557797_a_at,203603_s_at 229067_at 213692_s_at 227957_at 213296_at 235534_at233551_at 1558019_at

TABLE 3 matched term description 208073_x_at TTC3: tetratricopeptiderepeat domain 3 210645_s_at TTC3: tetratricopeptide repeat domain 3206630_at TYR: tyrosinase (oculocutaneous albinism IA) 203544_s_at STAM:signal transducing adaptor molecule (SH3 domain and ITAM motif) 1230741_at —: Full length insert cDNA clone YX74D05

TABLE 4 matched term description 205694_at TYRP1: tyrosinase-relatedprotein 1 206427_s_at MLANA: melan-A 206140_at LHX2: LIM homeobox 2206630_at TYR: tyrosinase (oculocutaneous albinism IA) 203921_at CHST2:carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 205337_at DCT:dopachrome tautomerase (dopachrome delta-isomerase, tyrosine-relatedprotein 2) 228245_s_at OVOS2: ovostatin 2 /// similar to cDNA sequenceBC048546 205338_s_at DCT: dopachrome tautomerase (dopachromedelta-isomerase, tyrosine-related protein 2) 1557797_a_at ZFHX1B: Zincfinger homeobox 1b 204271_s_at EDNRB: endothelin receptor type B237070_at TRPM1: transient receptor potential cation channel, subfamilyM, member 1 200716_x_at RPL13A: ribosomal protein L13a 1555579_s_atPTPRM: protein tyrosine phosphatase, receptor type, M 205051_s_at KIT:v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog200665_s_at SPARC: secreted protein, acidic, cysteine-rich (osteonectin)/// secreted protein, acidic, cysteine-rich (osteonectin) 205174_s_atQPCT: glutaminyl-peptide cyclotransferase (glutaminyl cyclase)200725_x_at RPL10: ribosomal protein L10 232602_at WFDC3: WAPfour-disulfide core domain 3 202478_at TRIB2: tribbles homolog 2(Drosophila) 209230_s_at P8: p8 protein (candidate of metastasis 1)232676_x_at MYEF2: myelin expression factor 2 222565_s_at PRKD3: proteinkinase D3 212295_s_at SLC7A1: solute carrier family 7 (cationic aminoacid transporter, y+ system), member 1 212594_at PDCD4: programmed celldeath 4 (neoplastic transformation inhibitor) 218211_s_at MLPH:melanophilin 206426_at MLANA: melan-A 207065_at K6HF: cytokeratin typeII 202500_at DNAJB2: DnaJ (Hsp40) homolog, subfamily B, member 2203706_s_at FZD7: frizzled homolog 7 (Drosophila) 209969_s_at STAT1:signal transducer and activator of transcription 1, 91 kDa

TABLE 5 matched term description 205694_at tyrosinase-related protein 1206140_at LIM homeobox 2 206427_s_at melan-A 203455_s_atspermidine/spermine N1-acetyltransferase 206453_s_at NDRG family member2 203921_at carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2200958_s_at syndecan binding protein (syntenin) 209283_at crystallin,alpha B 204271_s_at endothelin receptor type B 208073_x_attetratricopeptide repeat domain 3 232602_at WAP four-disulfide coredomain 3 202435_s_at cytochrome P450, family 1, subfamily B, polypeptide1 209230_s_at p8 protein (candidate of metastasis 1) 208966_x_atinterferon, gamma-inducible protein 16 205337_at dopachrome tautomerase(dopachrome delta-isomerase, tyrosine-related protein 2) 202088_atsolute carrier family 39 (zinc transporter), member 6 211538_s_at heatshock 70 kDa protein 2 201556_s_at vesicle-associated membrane protein 2(synaptobrevin 2) 241455_at Similar to AI661453 protein 237070_attransient receptor potential cation channel, subfamily M, member 1

TABLE 6 matched term description 1555505_a_at tyrosinase (oculocutaneousalbinism IA) 204271_s_at endothelin receptor type B 208073_x_attetratricopeptide repeat domain 3 200958_s_at syndecan binding protein(syntenin) 205051_s_at v-kit Hardy-Zuckerman 4 feline sarcoma viraloncogene homolog 201245_s_at OTU domain, ubiquitin aldehyde binding 1201603_at protein phosphatase 1, regulatory (inhibitor) subunit 12A201605_x_at calponin 2 201908_at dishevelled, dsh homolog 3 (Drosophila)202478_at tribbles homolog 2 (Drosophila) 1557292_a_at mucolipin 3200601_at actinin, alpha 4 200819_s_at ribosomal protein S15 209953_s_atCDC37 cell division cycle 37 homolog (S. cerevisiae) 213146_at jumonjidomain containing 3 222670_s_at v-maf musculoaponeurotic fibrosarcomaoncogene homolog B (avian) 224991_at c-Maf-inducing protein 226988_s_atmyosin, heavy polypeptide 14 244829_at Hypothetical protein MGC40222

TABLE 7 matched term description 204271_s_at endothelin receptor type B244829_at Hypothetical protein MGC40222 208073_x_at tetratricopeptiderepeat domain 3 213037_x_at staufen, RNA binding protein (Drosophila)200601_at actinin, alpha 4 219387_at KIAA1212 209168_at glycoprotein M6B205051_s_at v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogenehomolog 224991_at c-Maf-inducing protein 200613_at adaptor-relatedprotein complex 2, mu 1 subunit 203330_s_at syntaxin 5A 225009_atchemokine-like factor superfamily 4 221485_at UDP-Gal:betaGlcNAc beta1,4-galactosyltransferase, polypeptide 5 218255_s_at fibrosin 1227870_at likely ortholog of mouse neighbor of Punc E11 226988_s_atmyosin, heavy polypeptide 14 204086_at preferentially expressed antigenin melanoma 213146_at jumonji domain containing 3 205681_at BCL2-relatedprotein A1 213940_s_at formin binding protein 1 202478_at tribbleshomolog 2 (Drosophila) 226702_at hypothetical protein LOC129607218402_s_at Hermansky-Pudlak syndrome 4 227099_s_at hypotheticalLOC387763 218211_s_at melanophilin 217738_at pre-B-cell colony enhancingfactor 1 228488_at TBC1 domain family, member 16 215695_s_at glycogenin2 241898_at Transcribed locus, moderately similar to XP_517655.1PREDICTED: similar to KIAA0825 protein [Pan troglodytes] 202479_s_attribbles homolog 2 (Drosophila) 201453_x_at Ras homolog enriched inbrain 228415_at Adaptor-related protein complex 1, sigma 2 subunit201908_at dishevelled, dsh homolog 3 (Drosophila) 225600_at MRNA; cDNADKFZp779L1068 (from clone DKFZp779L1068) 221951_at transmembrane protein80 203455_s_at spermidine/spermine N1-acetyltransferase 201603_atprotein phosphatase 1, regulatory (inhibitor) subunit 12A 1558702_atTestis expressed sequence 10 204527_at myosin VA (heavy polypeptide 12,myoxin) 235222_x_at baculoviral IAP repeat-containing 4 1560445_x_at Rhoguanine nucleotide exchange factor (GEF) 1 1556205_at CDNA FLJ37227 fis,clone BRAMY2000277 226054_at bromodomain containing 4 210198_s_atproteolipid protein 1 (Pelizaeus-Merzbacher disease, spastic paraplegia2, uncomplicated) 202370_s_at core-binding factor, beta subunit209058_at endothelial differentiation-related factor 1 211755_s_at ATPsynthase, H+ transporting, mitochondrial F0 complex, subunit b, isoform1; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit b,isoform 1 229713_at CDNA FLJ13267 fis, clone OVARC1000964 209514_s_atRAB27A, member RAS oncogene family 201299_s_at MOB1, Mps One Binderkinase activator-like 1B (yeast) 211909_x_at prostaglandin E receptor 3(subtype EP3); prostaglandin E receptor 3 (subtype EP3) 209234_atkinesin family member 1B 207622_s_at ATP-binding cassette, sub-family F(GCN20), member 2 212421_at chromosome 22 open reading frame 9219636_s_at armadillo repeat containing 9 223407_at chromosome 16 openreading frame 48 200645_at GABA(A) receptor-associated protein242049_s_at neuroblastoma-amplified protein 230793_at Leucine richrepeat containing 16 215409_at PLSC domain containing protein202984_s_at BCL2-associated athanogene 5 201864_at GDP dissociationinhibitor 1 209780_at putative homeodomain transcription factor 2218143_s_at secretory carrier membrane protein 2 228919_at 228095_at PHDfinger protein 14 213736_at Cytochrome c oxidase subunit Vb 213655_atTyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,epsilon polypeptide 218419_s_at hypothetical protein MGC3123 200755_s_atcalumenin 223220_s_at poly (ADP-ribose) polymerase family, member 9237464_at LAT1-3TM protein 2 229679_at FLJ40142 protein IL-1 RI(Interleukin-1 RI) EDN2 (endothelin-2) EFNA5 (ephrin-A5) IGFBP7 (IGFBinding Protein 7) HLA-A0202 heavy chain (Human Leukocyte Antigen-A0202heavy chain) Activin A (βA subunit) TNF RII (tumor necrosis factorreceptor II) SPC4 (Subtilisin-Like Proprotein Convertase, PACE4) CNTF Rα(Ciliary neurotrophic factor receptor α)

TABLE 8 Gene Description 204271_s_at endothelin receptor type B244829_at Hypothetical protein MGC40222 208073_x_at tetratricopeptiderepeat domain 3 213037_x_at staufen, RNA binding protein (Drosophila)200601_at actinin, alpha 4 219387_at KIAA1212 209168_at glycoprotein M6B205051_s_at v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogenehomolog 224991_at c-Maf-inducing protein 200613_at adaptor-relatedprotein complex 2, mu 1 subunit 203330_s_at syntaxin 5A 225009_atchemokine-like factor superfamily 4 221485_at UDP-Gal:betaGlcNAc beta1,4-galactosyltransferase, polypeptide 5 218255_s_at fibrosin 1227870_at likely ortholog of mouse neighbor of Punc E11 226988_s_atmyosin, heavy polypeptide 14 204086_at preferentially expressed antigenin melanoma 213146_at jumonji domain containing 3 205681_at BCL2-relatedprotein A1 213940_s_at formin binding protein 1

TABLE 10 Gene Description 221750_at 3-hydroxy-3-methylglutaryl-CoenzymeA synthase 1 (soluble) 225283_at arrestin domain containing 4 212952_atCalreticulin 226920_at Casein kinase 1, alpha 1 201533_at catenin(cadherin-associated protein), beta 1, 88 kDa 225551_at chromosome 1open reading frame 71 227736_at chromosome 10 open reading frame 99217883_at chromosome 2 open reading frame 25 226614_s_at chromosome 8open reading frame 13 214073_at cortactin 233929_x_at CXYorf1-relatedprotein 225035_x_at CXYorf1-related protein; CXYorf1-related protein;CXYorf1-related protein 200953_s_at cyclin D2 206595_at cystatin E/M224831_at cytoplasmic polyadenylation element binding protein 4201211_s_at DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked 200762_atdihydropyrimidinase-like 2 219648_at dilute suppressor 202572_s_atdiscs, large (Drosophila) homolog-associated protein 4 200664_s_at DnaJ(Hsp40) homolog, subfamily B, member 1 208811_s_at DnaJ (Hsp40) homolog,subfamily B, member 6 208370_s_at Down syndrome critical region gene 1214445_at elongation factor, RNA polymerase II, 2 214446_at elongationfactor, RNA polymerase II, 2 201436_at eukaryotic translation initiationfactor 4E 208290_s_at eukaryotic translation initiation factor 5200748_s_at ferritin, heavy polypeptide 1 211628_x_at ferritin, heavypolypeptide pseudogene 1; ferritin, heavy polypeptide pseudogene 1205409_at FOS-like antigen 2 200959_at fusion (involved in t(12; 16) inmalignant liposarcoma) 201065_s_at general transcription factor II, i;general transcription factor II, i, pseudogene 1 218238_at GTP bindingprotein 4 201841_s_at heat shock 27 kDa protein 1 225988_at hect domainand RLD 4 241683_at HECT domain containing 1 201944_at hexosaminidase B(beta polypeptide) 219976_at hook homolog 1 (Drosophila) 213079_athypothetical protein DT1P1A10 215434_x_at hypothetical protein FLJ20719;AG1 protein 1569157_s_at hypothetical protein LOC162993 227052_atHypothetical protein LOC201895 225065_x_at hypothetical protein MGC40157231733_at ICEBERG caspase-1 inhibitor 240941_at Intersectin 2208881_x_at isopentenyl-diphosphate delta isomerase 1 204615_x_atisopentenyl-diphosphate delta isomerase 1 213507_s_at karyopherin(importin) beta 1 203068_at kelch-like 21 (Drosophila) 225142_atKIAA1718 protein 220368_s_at KIAA2010 1559226_x_at late cornifiedenvelope 1E 1559224_at late cornified envelope 1E 200673_atlysosomal-associated protein transmembrane 4 alpha 223480_s_atmitochondrial ribosomal protein L47 207121_s_at mitogen-activatedprotein kinase 6 214939_x_at myeloid/lymphoid or mixed-lineage leukemia(trithorax homolog, Drosophila); translocated to, 4 203315_at NCKadaptor protein 2 230291_s_at Nuclear factor I/B 211467_s_at nuclearfactor I/B 213032_at Nuclear factor I/B 223650_s_at nuclear receptorbinding factor 2 222878_s_at OTU domain, ubiquitin aldehyde binding 2217608_at p18 splicing regulatory protein 200907_s_at palladin 202290_atPDGFA associated protein 1 218942_at phosphatidylinositol-4-phosphate5-kinase, type II, gamma 225147_at pleckstrin homology, Sec7 andcoiled-coil domains 3 216515_x_at prothymosin, alpha (gene sequence 28);hypothetical gene supported by BC013859; hypothetical gene supported byBC013859; BC070480 200773_x_at prothymosin, alpha (gene sequence 28);similar to prothymosin alpha; hypothetical gene supported by BC013859;hypothetical gene supported by BC013859; BC070480 212099_at ras homologgene family, member B 212124_at retinoic acid induced 17 200022_atribosomal protein L18; ribosomal protein L18 201909_at ribosomal proteinS4, Y-linked 1 215127_s_at RNA binding motif, single strandedinteracting protein 1 218143_s_at secretory carrier membrane protein 2205185_at serine peptidase inhibitor, Kazal type 5 1554089_s_atShwachman-Bodian-Diamond syndrome; Shwachman-Bodian-Diamond syndromepseudogene 208991_at signal transducer and activator of transcription 3(acute-phase response factor) 224573_at similar to DNA segment, Chr 11,Brigham & Womens Genetics 0434 expressed 242687_at Similar to RIKEN cDNA9930021J17 206675_s_at SKI-like 1553602_at small breast epithelial mucin213879_at SMT3 suppressor of mif two 3 homolog 2 (yeast) 208738_x_atSMT3 suppressor of mif two 3 homolog 2 (yeast); similar to SMT3suppressor of mif two 3 homolog 2 1556839_s_at Spectrin, beta,non-erythrocytic 5 220983_s_at sprouty homolog 4 (Drosophila); sproutyhomolog 4 (Drosophila) 205966_at TAF13 RNA polymerase II, TATA boxbinding protein (TBP)-associated factor, 18 kDa 217733_s_at thymosin,beta 10 216438_s_at thymosin, beta 4, X-linked; thymosin-like 3226835_s_at transaldolase 1; similar to RPE-spondin 224680_attransmembrane emp24 protein transport domain containing 4 210987_x_attropomyosin 1 (alpha) 211702_s_at ubiquitin specific peptidase 32;ubiquitin specific peptidase 32 203798_s_at visinin-like 1 210935_s_atWD repeat domain 1 224905_at WD repeat domain 26 215150_at YOD1 OTUdeubiquinating enzyme 1 homolog (yeast) 227309_at YOD1 OTUdeubiquinating enzyme 1 homolog (yeast) 204180_s_at zinc finger protein297B 219163_at zinc finger protein 562 220854_at 224051_at 224050_s_at

TABLE 11 Gene Description 225519_at protein phosphatase 4, regulatorysubunit 2 219199_at AF4/FMR2 family, member 4 203450_at PKD2 interactor,golgi and endoplasmic reticulum associated 1 213729_at formin bindingprotein 3 220748_s_at zinc finger protein 580 216480_x_atMyeloid/lymphoid or mixed-lineage leukemia (trithorax homolog,Drosophila); translocated to, 10 200043_at enhancer of rudimentaryhomolog (Drosophila); enhancer of rudimentary homolog (Drosophila)211075_s_at CD47 antigen (Rh-related antigen, integrin-associated signaltransducer); CD47 antigen (Rh-related antigen, integrin-associatedsignal transducer) 1555945_s_at chromosome 9 open reading frame 10212295_s_at solute carrier family 7 (cationic amino acid transporter, y+system), member 1 212687_at LIM and senescent cell antigen-like domains1 224714_at MKI67 (FHA domain) interacting nucleolar phosphoprotein218768_at nucleoporin 107 kDa 228196_s_at La ribonucleoprotein domainfamily, member 5 217836_s_at YY1 associated protein 1 212620_at zincfinger protein 609 226845_s_at myeloma overexpressed 2 200747_s_atnuclear mitotic apparatus protein 1 242304_at within bgcn homolog(Drosophila) 204767_s_at flap structure-specific endonuclease 1217869_at hydroxysteroid (17-beta) dehydrogenase 12 222729_at F-box andWD-40 domain protein 7 (archipelago homolog, Drosophila) 201776_s_atKIAA0494 1552658_a_at neuron navigator 3 1555972_s_at F-box protein 28216242_x_at DNA directed RNA polymerase II polypeptide J-related gene231505_s_at Sideroflexin 4 228738_at hypothetical protein MGC25181228942_s_at DAB2 interacting protein 208959_s_at thioredoxin domaincontaining 4 (endoplasmic reticulum) 223407_at chromosome 16 openreading frame 48 1555278_a_at cytoskeleton associated protein 5219375_at choline/ethanolamine phosphotransferase 1 208728_s_at celldivision cycle 42 (GTP binding protein, 25 kDa) 50376_at zinc fingerprotein 444 243108_at RAN binding protein 9 212884_x_at Apolipoprotein E65630_at transmembrane protein 80 214953_s_at amyloid beta (A4)precursor protein (peptidase nexin-II, Alzheimer disease) 223946_atcofactor required for Sp1 transcriptional activation, subunit 3, 130 kDa232926_x_at ankyrin repeat domain 19 203597_s_at WW domain bindingprotein 4 (formin binding protein 21) 223601_at olfactomedin 2 212365_atmyosin IB 203297_s_at Jumonji, AT rich interactive domain 2 231019_x_atSerine/threonine kinase 11 (Peutz-Jeghers syndrome) 201291_s_attopoisomerase (DNA) II alpha 170 kDa 211846_s_at poliovirusreceptor-related 1 (herpesvirus entry mediator C; nectin) 226843_s_atPAP associated domain containing 5 225243_s_at sarcolemma associatedprotein 236651_at kalirin, RhoGEF kinase 214792_x_at vesicle-associatedmembrane protein 2 (synaptobrevin 2) 228922_at Src homology 2 domaincontaining F 225537_at trafficking protein particle complex 6B 46665_atsema domain, immunoglobulin domain (Ig), transmembrane domain (TM) andshort cytoplasmic domain, (semaphorin) 4C 209702_at fatso 203358_s_atenhancer of zeste homolog 2 (Drosophila) 211310_at enhancer of zestehomolog 1 (Drosophila) 242767_at LIM and cysteine-rich domains 11555575_a_at KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum proteinretention receptor 1 223151_at DCN1, defective in cullin neddylation 1,domain containing 5 (S. cerevisiae) 204170_s_at CDC28 protein kinaseregulatory subunit 2 229420_at Luminal binding protein 1 (BiP-1) (BP1)202355_s_at general transcription factor IIF, polypeptide 1, 74 kDa206061_s_at Dicer1, Dcr-1 homolog (Drosophila) 224597_at Transcribedlocus, strongly similar to XP_523650.1 PREDICTED: similar to keratin 17[Pan troglodytes] 217739_s_at pre-B-cell colony enhancing factor 1218943_s_at DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 211087_x_atmitogen-activated protein kinase 14; mitogen-activated protein kinase 14220193_at chromosome 1 open reading frame 113 229410_atprogestagen-associated endometrial protein (placental protein 14,pregnancy-associated endometrial alpha-2-globulin, alpha uterineprotein) 221844_x_at CDNA clone IMAGE: 6208446 227683_x_at Nudix(nucleoside diphosphate linked moiety X)-type motif 4 pseudogene 2233621_s_at Rho guanine nucleotide exchange factor (GEF) 12 214270_s_atmicrotubule-associated protein, RP/EB family, member 3 217762_s_atRAB31, member RAS oncogene family 231271_x_at HSCARG protein 227330_x_atsimilar to hypothetical protein MGC27019 209773_s_at ribonucleotidereductase M2 polypeptide 225227_at SKI-like 218428_s_at REV1-like(yeast) 201556_s_at vesicle-associated membrane protein 2 (synaptobrevin2)

TABLE 12 Gene Description 1552477_a_at interferon regulatory factor 6228707_at claudin 23 206427_s_at melan-A 218196_at osteopetrosisassociated transmembrane protein 1 219142_at RAS-like, family 11, memberB 200601_at actinin, alpha 4 226483_at transmembrane protein 68243568_at Glycine-rich protein (GRP3S) 212382_at Transcription factor 4218417_s_at hypothetical protein FLJ20489 208905_at cytochrome c,somatic 203753_at transcription factor 4 244535_at Forkhead box P1222243_s_at transducer of ERBB2, 2 205174_s_at glutaminyl-peptidecyclotransferase (glutaminyl cyclase) 231851_at hypothetical proteinFLJ10770 200961_at selenophosphate synthetase 2 210880_s_at embryonalFyn-associated substrate 230986_at Kruppel-like factor 8 229689_s_atDiscs, large homolog 5 (Drosophila) 204319_s_at regulator of G-proteinsignalling 10 219842_at ADP-ribosylation factor related protein 2224560_at TIMP metallopeptidase inhibitor 2 208758_at5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMPcyclohydrolase 238662_at similar to RIKEN cDNA 5730421E18 gene214000_s_at Regulator of G-protein signalling 10 1559360_at NuclearRNA-binding protein, putative 205694_at tyrosinase-related protein 1231579_s_at TIMP metallopeptidase inhibitor 2 238967_at Claudin 1222146_s_at transcription factor 4 230748_at solute carrier family 16(monocarboxylic acid transporters), member 6; similar to solute carrierfamily 16, member 6; monocarboxylate transporter 6

TABLE 13 Sample Melanoma Melanoma Melanoma Melanoma Melanoma Melanomagene description DT357-M DT330-M DT359-M DT419-M DT407-M DT412-M244829_at C6orf218 114.5632 19.56224 0.594604 7.412704 53.81737 0.456916204271_s_at EDNRB 225.972 151.1671 18.89588 16.67945 754.8258 17.26765200601_at ACTN4 30.90996 23.10287 10.05611 1.484524 60.96883 5.241574226988_s_at MYH14 0.192109 0.343885 0.032804 0.140632 0.009486 0.554785202478_at TRIB2 99.73307 75.58353 28.84001 12.72858 464.6498 15.670721557292_a_at MCOLN3 28.05138 8.282119 5.464161 4.084049 64.893413.555371 224991_at CMIP 0.615572 0.208772 0.214641 0.205898 0.1150231.741101 1555505_a_at TYR 21.25897 23.26356 9.849155 0.952638 150.12295.426417 201908_at DVL3 0.339151 0.088388 0.239816 8.111676 5.5021674.924578 222670_s_at MAFB 0.070316 0.069348 0.005263 0.00146 0.5321850.164938 201605_x_at CNN2 150.1229 58.48521 15.13692 13.54793 324.033717.5087 213146_at JMJD3 0.005048 0.003879 0.02683 0.00198 0.0094860.001211 201603_at PPP1R12A 2.188587 4.69134 0.817902 0.281265 7.3615010.05954 209953_s_at CDC37 12.72858 34.05985 4.756828 0.615572 13.737052.732081 201245_s_at OTUB1 9.063071 6.233317 5.464161 1.905276 10.777871.580083 208073_x_at TTC3 15.34823 12.72858 8.168097 8.224911 36.504446.916298 200958_s_at SDCBP 3.07375 1.197479 0.336808 0.078021 6.9162980.065154 205051_s_at KIT 86.82268 54.1917 2.713209 8.815241 55.715244.055838 200819_s_at RPS15 1584.707 1640.591 491.1432 182.2784 3795.305377.4129 Sample Melanoma Melanoma Melanoma Melanoma gene descriptionDT403-M DT406-M DT356-M DT405-M 244829_at C6orf218 16.44982 10884.5917.14838 2797.65 204271_s_at EDNRB 81.00842 45073.75 63.11889 11828.67200601_at ACTN4 38.85424 4269.94 9.000468 13682.08 226988_s_at MYH140.004613 1 0.164938 1045.516 202478_at TRIB2 19.42712 1 24.9332714972.21 1557292_a_at MCOLN3 3.24901 1067.485 4.756828 1082.386224991_at CMIP 1.148698 1 0.122428 1351.176 1555505_a_at TYR 2.9281714870.992 2.732081 3468.269 201908_at DVL3 1.292353 32.89964 4.1698637281.399 222670_s_at MAFB 0.145592 1 0.000816 103.9683 201605_x_at CNN228.44297 22226.61 32.67239 46340.95 213146_at JMJD3 0.018326 37.014020.078563 50.91433 201603_at PPP1R12A 0.926588 481.0356 0.103665 205.0739209953_s_at CDC37 21.70567 1226.218 7.310652 9410.137 201245_s_at OTUB111.87619 3691.522 1.840375 4182.066 208073_x_at TTC3 7.674113 4973.3421.741101 4299.64 200958_s_at SDCBP 0.389582 261.3791 0.5 247.2797205051_s_at KIT 0.435275 3082.745 12.81712 689.7836 200819_s_at RPS151884.544 389158.9 484.3815 668236.8

TABLE 14 Sample Nevus Nevus Nevus Nevus Nevus gene description DF543DF544 DT343 DT342 DT344 244829_at C6orf218 0.094732 0.80107 0.2973020.00849 0.000905 204271_s_at EDNRB 393.44 16 401.7071 0.135842 0.010598200601_at ACTN4 0.289172 44.3235 5712.87 65.79928 12.21007 226988_s_atMYH14 0.094732 1.404445 430.539 48.50293 0.109576 202478_at TRIB223.26356 6.868523 797.8645 0.00849 0.005601 1557292_a_at MCOLN3 0.250.882703 0.297302 0.00849 0.000905 224991_at CMIP 3.944931 0.664343132.5139 7.727491 0.61132 1555505_a_at TYR 2.297397 9.189587 0.2973020.00849 0.010167 201908_at DVL3 0.094732 2.219139 139.1021 0.9930920.248273 222670_s_at MAFB 0.233258 1.006956 41.93259 1.879045 0.112656201605_x_at CNN2 41.93259 183.5463 21027.65 855.13 50.91433 213146_atJMJD3 0.094732 0.496546 4.69134 0.07911 0.000905 201603_at PPP1R12A0.094732 3.680751 85.03589 19.02731 0.543367 209953_s_at CDC37 1.9185288.815241 699.4126 34.5353 0.959264 201245_s_at OTUB1 0.094732 11.876191663.493 45.25483 11.08088 208073_x_at TTC3 0.882703 58.08123 1478.58312.295 0.132127 200958_s_at SDCBP 0.094732 0.188156 3.317278 0.9862330.016402 205051_s_at KIT 0.094732 0.239816 34.5353 2.602684 0.011203200819_s_at RPS15 13124.73 4299.64 205674 11346.82 584.071 Sample NevusNevus Nevus Nevus Nevus gene description DT345 DT427 DT337 DT340 DT338244829_at C6orf218 1 0.550953 0.939523 0.001236 0.479632 204271_s_atEDNRB 225.972 1136.199 0.20733 0.001236 393.44 200601_at ACTN4 14462.213350.127 127.1158 64.44516 3236.009 226988_s_at MYH14 2304.12 867.06722.907945 1.265757 410.1478 202478_at TRIB2 1 8422.308 4.287094 3.43426212.46663 1557292_a_at MCOLN3 1 116.9704 14.12325 0.001236 0.479632224991_at CMIP 1686.714 369.6459 1.021012 1.918528 433.5336 1555505_a_atTYR 63.55792 10.33882 1.94531 0.001236 39.67065 201908_at DVL3 404.50121097.496 6.588728 1.580083 9.38268 222670_s_at MAFB 418.7659 12.640660.946058 0.002577 51.98415 201605_x_at CNN2 3615.551 10015.87 181.019358.48521 3821.703 213146_at JMJD3 1.580083 0.550953 0.628507 0.0012360.479632 201603_at PPP1R12A 16.22335 202.2506 3.160165 0.008373 942.2722209953_s_at CDC37 2957.167 94.35323 60.54769 7.110741 3929.146201245_s_at OTUB1 9026.807 10297.45 47.50475 9.063071 2179.83208073_x_at TTC3 6472.018 1937.526 50.56264 0.314253 2836.704200958_s_at SDCBP 7.568461 0.550953 0.346277 0.001236 1.049717205051_s_at KIT 1 37.01402 1.22264 0.001236 0.479632 200819_s_at RPS15736333.6 137588.5 7967.989 600.4915 269513.9

TABLE 15 (Lentigo Maligna)/(Solar Lentigo Maligna Solar lentigo Lentigo)Gene Mean expression Mean expression fold change Description 200961_at455.88 223.03 2.04 selenophosphate synthetase 2 200782_at 379.88 70.685.37 annexin A5 206427_s_at 1899.38 165.82 11.45 melan-A 217998_at416.81 99.94 4.17 pleckstrin homology-like domain, family A, member 1226602_s_at 117.73 209.24 0.56 breakpoint cluster region; similar tobreakpoint cluster region isoform 1 240366_at 70.62 6.65 10.62 LipomaHMGIC fusion partner-like 3 208325_s_at 760.50 1233.35 0.62 A kinase(PRKA) anchor protein 13 225202_at 196.73 24.59 8.00 Rho-related BTBdomain containing 3 225946_at 46.81 5.74 8.16 Ras association(RalGDS/AF-6) domain family 8 1553603_s_at 37.15 61.18 0.61ADP-ribosylation factor-like 6 interacting protein 2 220625_s_at 125.2375.62 1.66 E74-like factor 5 (ets domain transcription factor) 229982_at28.00 20.79 1.35 hypothetical protein FLJ21924 1552283_s_at 17.50 35.560.49 zinc finger, DHHC-type containing 11 200723_s_at 203.23 113.68 1.79membrane component, chromosome 11, surface marker 1 209174_s_at 57.35106.44 0.54 FLJ20259 protein 233599_at 244.31 403.97 0.60 Chromosome 9open reading frame 3 201739_at 4791.23 2597.32 1.84 serum/glucocorticoidregulated kinase 209392_at 403.54 12.79 31.54 ectonucleotidepyrophosphatase/phosphodiesterase 2 (autotaxin) 209487_at 185.54 46.444.00 RNA binding protein with multiple splicing 221653_x_at 882.08458.24 1.92 apolipoprotein L, 2 209185_s_at 349.73 118.85 2.94 insulinreceptor substrate 2 222809_x_at 227.73 336.12 0.68 chromosome 14 openreading frame 65 223363_at 150.69 280.06 0.54 hypothetical proteinMGC10911 208456_s_at 56.19 122.65 0.46 related RAS viral (r-ras)oncogene homolog 2 221449_s_at 69.81 41.09 1.70 T-cell immunomodulatoryprotein; T-cell immunomodulatory protein 215268_at 24.12 46.74 0.52KIAA0754 protein 217188_s_at 146.88 397.50 0.37 chromosome 14 openreading frame 1 236972_at 302.00 27.09 11.15 tripartite motif-containing63

TABLE 16 solar solar solar solar solar solar solar solar solar solarsolar solar lentigo lentigo lentigo lentigo lentigo lentigo lentigolentigo lentigo lentigo lentigo lentigo DF639- DF640- DF641- DF642- geneDF529-S DF530-S DF633-S DF634-S DF635-S DF636-S DF637-S DF638-S S S S S200961_at 106 217 101 38 161 221 160 336 304 256 63 210 200782_at 122 2145 8 70 27 104 195 200 63 101 114 206427_s_at 58 28 92 19 132 29 92 721708 12 92 190 217998_at 106 67 180 18 86 76 181 95 238 169 7 8226602_s_at 654 97 144 691 129 265 109 194 137 277 194 130 240366_at 6 67 6 5 6 27 6 6 6 6 6 225202_at 5 6 22 9 84 14 13 14 35 8 21 5 225946_at5 5 5 5 6 5 5 5 13 6 5 5 1553603_s_at 33 70 60 5 67 59 40 26 54 127 2143 220625_s_at 37 35 6 6 17 173 55 29 42 5 13 5 229982_at 58 17 25 9 1513 22 17 9 33 16 12 1552283_s_at 14 31 38 14 49 10 14 33 19 23 41 13200723_s_at 94 159 201 22 263 94 299 157 95 217 75 93 209174_s_at 118143 64 25 107 89 103 236 46 256 38 110 233599_at 89 322 294 116 433 380295 370 306 241 252 294 201739_at 1421 958 3724 618 2023 2741 3098 40623856 1907 1530 1569 209392_at 7 44 6 19 10 9 9 10 47 6 17 17 209487_at 6403 9 6 16 6 24 163 94 5 5 23 221653_x_at 88 379 298 349 396 575 322 3723571 319 1159 1349 209185_s_at 307 365 182 58 86 140 195 127 44 97 165130 222809_x_at 646 253 322 6 358 262 319 316 490 277 381 654 223363_at206 112 192 102 324 318 343 323 185 694 706 860 208456_s_at 322 22 30 3483 22 125 71 129 125 43 122 221449_s_at 35 26 73 7 69 31 51 37 8 52 6649 215268_at 11 12 43 9 30 8 22 8 44 73 11 51 217188_s_at 1276 36 545 12519 479 281 801 209 500 99 457 236972_at 6 6 6 11 6 6 6 6 12 6 6 13

TABLE 17 solar solar solar solar solar solar solar solar solar solarsolar solar lentigo lentigo lentigo lentigo lentigo lentigo lentigolentigo lentigo lentigo lentigo lentigo DT306- DT365- DT367- DT368- geneDF643-S DT024-S DT055-S DT069-S DT079-S DT123-S DT146-S DT187-S S S S S200961_at 223 149 159 8 113 202 2155 152 177 56 76 180 200782_at 17 79 684 136 8 5 95 65 34 29 11 206427_s_at 9 444 5 13 29 7 11 500 672 5 6 20217998_at 15 178 8 104 181 64 6 62 31 205 42 16 226602_s_at 286 212 32797 368 456 57 84 154 301 50 24 240366_at 6 6 6 7 6 6 6 6 6 6 6 6208325_s_at 1565 923 877 30 973 1508 1981 1326 1289 863 354 3194225202_at 49 40 8 9 21 101 8 47 22 12 31 8 225946_at 5 6 5 6 6 5 5 5 9 55 6 1553603_s_at 54 72 76 21 84 84 44 71 80 71 100 168 220625_s_at 118 56 5 5 14 865 17 5 5 6 6 229982_at 8 8 13 8 33 6 16 18 22 13 13 181552283_s_at 10 23 9 11 12 9 13 155 122 10 13 67 200723_s_at 32 21 21 7179 50 220 171 93 92 17 83 209174_s_at 172 38 48 85 256 115 110 147 109141 27 96 233599_at 594 125 489 243 695 36 931 591 623 1648 346 1044201739_at 3963 4104 8062 2767 2729 2760 378 2524 1236 3566 2237 785209392_at 8 8 7 10 10 7 7 35 14 7 9 7 209487_at 39 139 6 7 84 12 201 448 19 6 6 221653_x_at 330 198 139 243 341 331 301 382 511 137 564 306209185_s_at 53 83 133 7 201 137 170 91 84 78 32 86 222809_x_at 323 478622 65 190 729 938 438 211 284 180 249 223363_at 196 291 109 85 163 290331 165 279 161 145 234 208456_s_at 43 211 509 472 81 24 298 32 69 19 75231 221449_s_at 121 78 31 6 39 10 46 74 56 60 6 31 215268_at 36 19 18 8102 119 104 133 24 14 108 27 217188_s_at 692 225 228 11 480 1467 195 178306 423 29 189 236972_at 6 34 6 17 5 6 6 6 6 6 7 6

TABLE 18 solar solar solar solar solar solar solar solar solar solarlentigo lentigo lentigo lentigo lentigo lentigo lentigo lentigo lentigolentigo gene DT369-S DT370-S DT371-S DT372-S DT373-S DT409-S DT414-SDT422-S DT459-S DT460-S 200961_at 55 39 38 124 262 37 881 60 128 136200782_at 139 50 248 17 73 48 117 11 53 8 206427_s_at 9 92 20 7 625 9585 19 7 20 217998_at 496 34 123 58 140 71 22 31 98 182 226602_s_at 12346 107 349 95 97 184 440 119 117 240366_at 6 6 6 6 6 6 6 6 6 6208325_s_at 1373 60 557 201 1500 2266 1545 1500 2099 839 225202_at 20 129 122 9 8 5 11 40 8 225946_at 6 6 6 6 6 5 5 6 6 5 1553603_s_at 57 101 2234 47 33 47 39 102 68 220625_s_at 5 6 5 6 6 5 974 6 11 67 229982_at 58 613 13 13 8 13 31 117 13 1552283_s_at 65 12 18 9 15 6 87 138 25 81200723_s_at 95 97 11 41 97 77 253 7 103 329 209174_s_at 177 31 140 12328 163 134 13 47 84 233599_at 453 300 195 152 58 166 802 300 279 273201739_at 2723 3494 2757 850 3680 3553 721 897 3544 3472 209392_at 6 931 7 7 7 12 10 9 7 209487_at 15 6 7 6 6 6 166 7 23 6 221653_x_at 237 204353 362 354 249 229 108 171 353 209185_s_at 100 15 10 82 94 6 496 13 10173 222809_x_at 167 215 248 121 408 209 336 138 226 369 223363_at 121 14583 121 984 182 227 150 466 229 208456_s_at 168 41 216 39 230 22 77 23 28134 221449_s_at 34 6 5 10 7 160 18 7 27 61 215268_at 88 9 11 52 55 13 249 254 40 217188_s_at 647 119 93 79 122 694 269 111 834 910 236972_at 636 619 8 11 6 6 17 5 6

TABLE 19 lentigo lentigo lentigo lentigo lentigo lentigo lentigo lentigolentigo lentigo lentigo maligna maligna maligna maligna maligna malignamaligna maligna maligna maligna maligna DF569- DF557- DF579- DF580-DF582- DF596- DF623- DF624- DF625- DF626- DF627- gene LM LM LM LM LM LMLM LM LM LM LM 200961_at 847 567 158 531 234 359 357 330 347 518 1667200782_at 154 500 157 193 764 1310 331 27 157 161 16 206427_s_at 18332805 95 1327 2219 6320 3253 49 274 193 121 217998_at 185 1245 726 88 907773 789 182 340 114 10 226602_s_at 49 47 59 51 71 36 50 51 137 91 579240366_at 139 251 6 43 122 35 15 6 12 25 6 208325_s_at 20 1362 725 408882 430 568 1583 1867 1460 699 225202_at 25 16 44 312 237 1183 170 33 2683 8 225946_at 48 57 9 76 12 7 35 5 5 6 28 1553603_s_at 8 21 43 67 45 2137 38 50 23 10 220625_s_at 5 5 5 288 29 5 132 154 126 206 1427 229982_at8 8 19 8 28 21 13 44 35 28 93 1552283_s_at 11 14 12 16 5 13 68 10 35 1313 200723_s_at 159 480 276 85 369 112 349 90 319 242 134 209174_s_at 13524 48 68 87 130 40 58 85 110 60 233599_at 158 60 524 126 283 385 298 183228 276 165 201739_at 6073 10285 3008 5935 4757 3159 3792 3462 3128 2916826 209392_at 772 235 14 1088 210 243 912 9 8 8 7 209487_at 291 148 21315 280 1146 521 31 16 71 61 221653_x_at 433 203 613 743 1224 5336 983425 227 141 43 209185_s_at 1935 1458 124 482 85 535 182 98 169 308 154222809_x_at 33 32 55 45 161 31 36 437 163 170 1483 223363_at 161 136 13992 156 129 114 241 122 147 105 208456_s_at 20 27 27 21 49 57 8 33 39 21046 221449_s_at 7 7 28 54 30 50 199 185 57 24 156 215268_at 9 11 157 4117 11 42 11 27 9 9 217188_s_at 8 8 94 11 148 39 8 1146 455 62 107236972_at 1318 1977 6 137 77 247 51 8 31 6 13

TABLE 20 lentigo lentigo lentigo lentigo lentigo lentigo lentigo lentigolentigo lentigo lentigo lentigo maligna maligna maligna maligna malignamaligna maligna maligna maligna maligna maligna maligna DF629- DF630-DF631- DF632- DT017- DT266- DT268- DT269- DT270- DT331- DT355- DT423-gene LM LM LM LM LM LM LM LM LM LM LM LM 200961_at 350 139 465 196 510439 435 383 744 306 398 420 200782_at 291 156 265 8 551 52 255 33 825469 1914 308 206427_s_at 2834 317 1121 5 4173 290 1952 181 3873 39139382 990 217998_at 307 241 427 38 521 285 328 48 1470 344 879 184226602_s_at 114 46 230 74 48 184 81 280 13 46 30 107 240366_at 6 6 6 6 643 8 8 115 262 642 47 208325_s_at 529 101 448 1404 956 506 782 632 6082658 396 642 225202_at 139 152 36 66 143 121 83 46 1987 65 23 31225946_at 6 6 81 6 28 14 32 9 415 46 202 67 1553603_s_at 34 40 21 32 5213 37 37 22 56 11 37 220625_s_at 5 6 69 95 11 23 33 90 5 6 6 239229982_at 36 14 56 47 29 13 21 13 12 13 12 12 1552283_s_at 13 12 10 1016 10 13 9 38 26 34 13 200723_s_at 321 300 70 118 138 68 21 54 80 97 67113 209174_s_at 84 52 12 40 38 26 84 70 28 47 63 29 233599_at 296 32 9680 1338 115 314 86 131 497 62 235 201739_at 2421 3869 4568 3885 26794995 4927 4514 12929 5385 9033 5005 209392_at 273 223 464 11 656 26 4987 1581 287 2683 238 209487_at 101 6 338 6 661 19 36 6 366 33 243 90221653_x_at 626 873 983 336 1599 265 780 493 516 1548 1540 437209185_s_at 389 115 164 214 105 201 102 255 1272 161 139 160 222809_x_at166 105 461 83 125 527 435 453 22 186 56 322 223363_at 314 122 132 90191 105 206 373 167 113 167 104 208456_s_at 17 26 122 27 44 203 61 36 7122 27 196 221449_s_at 36 171 107 142 39 74 48 83 11 10 40 162 215268_at11 7 9 9 43 11 24 16 8 11 11 98 217188_s_at 175 638 24 79 138 166 95 508 144 25 84 236972_at 33 34 328 7 365 41 51 9 1766 698 421 200

TABLE 21 lentigo lentigo lentigo maligna maligna maligna DT425- DT461-DF523- gene LM LM LM 200961_at 798 348 7 200782_at 150 328 502206427_s_at 177 778 909 217998_at 38 233 135 226602_s_at 492 49 46240366_at 6 9 6 208325_s_at 20 38 49 225202_at 68 9 9 225946_at 5 6 61553603_s_at 168 21 22 220625_s_at 204 6 76 229982_at 58 62 251552283_s_at 13 18 10 200723_s_at 863 24 335 209174_s_at 49 12 12233599_at 93 53 238 201739_at 2997 4238 5786 209392_at 20 9 10 209487_at6 6 6 221653_x_at 750 1089 728 209185_s_at 273 7 6 222809_x_at 111 35188 223363_at 107 83 102 208456_s_at 22 10 40 221449_s_at 22 22 51215268_at 6 6 13 217188_s_at 38 6 63 236972_at 8 7 13

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A method for distinguishing melanoma fromnon-melanoma in a subject, the method comprising: (a) providing abiological sample of a skin lesion suspected of comprising melanoma fromthe subject; provided that the biological sample comprises a pluralityof gene products; and (b) determining an expression level of one or moregene products expressed from one or more genes of a gene classifiercomprising preferentially expressed antigen in melanoma (PRAME),C6orf218, or a combination thereof, in the biological sample; providedthat an increase in expression level of one or more of the one or moregene products expressed from the genes of the gene classifier in thebiological sample as compared to an expression level of thecorresponding gene products expressed from the one or more genes of thegene classifier in a non-melanoma sample is indicative of the skinlesion comprising melanoma.
 2. The method of claim 1, provided that anexpression level of a gene product expressed from PRAME is determined inthe biological sample; whereby an increase in the expression level ofthe gene product expressed from PRAME in the biological sample ascompared to an expression level of a gene product expressed from PRAMEin the non-melanoma skin sample is indicative of melanoma in thebiological sample.
 3. The method of claim 1, provided that an expressionlevel of a gene product expressed from PRAME is determined and providedthat an expression level of a gene product expressed from C6orf218 isdetermined in the biological sample; whereby an increase in expressionlevel of PRAME, C6orf218, or both PRAME and C6orf218 in the biologicalsample as compared to a corresponding expression level of PRAME,C6orf218, or both PRAME and C6orf218 in the non-melanoma sample isindicative of melanoma in the biological sample.
 4. The method of claim1, provided that the biological sample of the skin lesion is obtained byapplying an adhesive tape to the skin lesion in a manner sufficient toadhere a sample of the skin lesion to the adhesive tape, and removingthe adhesive tape from the skin lesion in a manner sufficient to retainthe adhered skin sample to the adhesive tape.
 5. The method of claim 4,provided that the tape comprises a rubber adhesive on a polyurethanefilm.
 6. The method of claim 5, provided that about one to about tenadhesive tapes or one to ten applications of the tape are applied to andremoved from the skin lesion.
 7. The method of claim 1, provided thatthe biological sample of the skin lesion is obtained by a biopsy of theskin lesion.
 8. The method of claim 1, provided that determining theexpression level of the gene products expressed from the one or moregenes of the gene classifier comprises applying to the biological sampleone or more detectably labeled probes that hybridize to the one or moregene products, amplification products of the one or more gene products,or a portion or combination thereof.
 9. The method of claim 1, providedthat the one or more gene products expressed form the one or more genesof the gene classifier comprise nucleic acid molecules.
 10. The methodof claim 9, provided that the nucleic acid molecules comprise RNAmolecules.
 11. The method of claim 10, provided that determining theexpression level of the gene products expressed from the one or moregenes of the gene classifier comprises generating cDNA molecules fromthe RNA molecules, and amplifying the cDNA molecules by real-timequantitative polymerase chain reaction (PCR).
 12. The method of claim 9,provided that determining the expression level of the gene productsexpressed from the one or more genes of the gene classifier comprisesmicroarray analysis, digital gene expression or a direct sequencingmethod.
 13. The method of claim 1, provided that the skin lesioncomprises melanoma, the method further comprising determining atreatment regimen.
 14. The method of claim 1, provided that theexpression level of the corresponding gene products in the non-melanomasample is contained within a database.
 15. The method of claim 14,provided that the expression level of the gene product in the biologicalsample is compared to the expression level of the corresponding geneproducts in the non-melanoma sample using a computer.
 16. A kit forcharacterizing a skin lesion in a subject comprising: a skin samplecollection device; and one or more primers that selectively bind to oneor more nucleic acid molecules expressed from PRAME, C6orf218, bothPRAME and C6orf218, or an amplification product or portion thereof. 17.The kit of claim 16, further comprising one or more detectably labeledprobes that selectively bind to one or more nucleic acid moleculesexpressed from PRAME, C6orf218, both PRAME and C6orf218, or anamplification product or portion thereof.
 18. The kit of claim 16,wherein the skin sample collection device comprises an adhesive tape.19. A method for distinguishing melanoma from non-melanoma in a subject,the method comprising: a) providing a biological sample comprising aplurality of nucleic acid molecules from a skin lesion of a subjectsuspected of comprising melanoma; b) determining an expression profileof one or more nucleic acid molecules expressed from one or more genesin a gene signature comprising PRAME, C6orf218, or a combinationthereof, by assaying the plurality of nucleic acid molecules in thebiological sample; c) comparing the expression profile of the biologicalsample to a threshold value; and d) classifying the lesion as comprisingmelanoma or not comprising melanoma based on the correlation between theexpression profile of the biological sample and the threshold value. 20.The method of claim 19, provided that the one or more nucleic acidmolecules expressed from the one or more genes in the gene signaturecomprise RNA molecules.
 21. The method of claim 20, provided thatassaying the plurality of nucleic acid molecules in the biologicalsample comprises generating complementary (cDNA) products from the RNAmolecules expressed from the one or more genes in the gene signature,and performing real-time quantitative PCR to amplify and quantify thecDNA products to determine expression levels of the one or more genes inthe gene signature.
 22. The method of claim 19, provided that thebiological sample of the skin lesion is obtained by applying an adhesivetape to the skin lesion in a manner sufficient to adhere a sample of theskin lesion to the adhesive tape, and removing the adhesive tape fromthe skin lesion in a manner sufficient to retain the adhered skin sampleto the adhesive tape.
 23. The method of claim 22, provided that the tapecomprises a rubber adhesive on a polyurethane film.
 24. The method ofclaim 22, provided that about one to about ten adhesive tapes or one toten applications of the tape are applied to and removed from the skinlesion.
 25. The method of claim 19, provided that the biological sampleof the skin lesion is obtained by a biopsy of the skin lesion.
 26. Themethod of claim 19, provided that assaying the plurality of nucleic acidmolecules in the biological sample comprises applying to the biologicalsample one or more detectably labeled probes that hybridize to a nucleicacid sequence from the one or more nucleic acid molecules expressed fromthe one or more genes in the gene signature, amplification productsthereof, or a combination thereof.
 27. The method of claim 19, providedthat assaying the plurality of nucleic acid molecules in the biologicalsample comprises microarray analysis, digital gene expression or adirect sequencing method.
 28. The method of claim 19, provided that theskin lesion comprises melanoma, the method further comprisingdetermining a treatment regimen.
 29. The method of claim 19, providedthat the threshold value is contained within a database.
 30. The methodof claim 29, provided that the expression profile is compared to thethreshold value using a computer.